Methods and tools for identifying compounds which modulate atherosclerosis by impacting LDL-proteoglycan binding

ABSTRACT

The present invention relates to the study and control of atherosclerosis through the modulation of LDL-proteoglycan binding at Site B (amino acids 3359-3369) of the apo-B100 protein in LDL. The invention encompasses methods of identifying compounds which modulate LDL-proteoglycan binding, methods of identifying compounds which modulate atherosclerotic lesion formation, and methods of modulating the formation of atherosclerotic lesions. The invention also encompasses mutant apo-B100 proteins and LDL which exhibit reduced proteoglycan binding while maintaining LDL-receptor binding, polynucleotides which encode these apo-B100 proteins, as well as cells and animals which express the mutant apo-B100 proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of application Ser. No.09/265,222, filed Mar. 5, 1999, now U.S. Pat. No. 6,579,682, whichclaims priority under 35 USC §119(e) to U.S. Provisional ApplicationSer. No. 60/077,618, filed Mar. 10, 1998, the entire contents of whichare incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

The invention was funded in part by National Institutes of Healthprogram project grant HL41633. The U.S. Government may have certainrights to this invention.

TECHNICAL FIELD

This invention relates to the disease atherosclerosis, methods ofmodulating the formation of atherosclerotic lesions, and methods ofidentifying compounds which modulate atherosclerotic lesion formation.Specifically the invention relates to the reduction of atherosclerosisthrough the modulation of LDL-proteoglycan binding at Site B (aminoacids 3359-3369) of the apo-B100 protein in LDL.

BACKGROUND ART

High levels of LDL are a major risk factor for coronary disease and arethe source for most of the cholesterol that accumulates in the arterialwall (Ross, R. 1995. Annu. Rev. Physiol. 57:791-804). Subendothelialretention of LDL has been suggested to be a key pathogenic process inatherosclerosis, and several lines of circumstantial evidence suggestthat intramural retention of atherogenic lipoproteins involves theextracellular matrix, chiefly proteoglycans (Hurt-Camejo, E. et al.1997. Arterioscler Thromb Vasc Biol. 17:1011-1017; Williams, K. J., andI. Tabas. 1995. Arterioscier. Thromb. Vasc. Biol. 15:551-561; andRadhakrishnamurthy, B. et al. 1990. Eur. Heart J. 11 Suppl E: 148-157).

The significance of the possible LDL proteoglycan interaction has beenhighlighted in two recent review articles (Hurt-Camejo, E. et al. 1997.Arterioscler Thromb Vasc Biol. 17:1011-1017; and Williams, K. J., and I.Tabas. 1995. Arterioscier. Thromb. Vasc Biol. 15:551-561). Williams andTabas proposed that subendothelial retention of atherogenic lipoproteinsis the central pathogenic process in atherosclerosis. Moreover, theyhypothesized that retained lipoproteins can directly or indirectlyprovoke all known features of early lesions, such as lipoproteinoxidation, monocyte migration into the artery wall, macrophage foam cellformation, and cytokine production, and can accelerate further retentionby stimulating local synthesis of proteoglycans. Several lines ofevidence indicate that the retention of arterial lipoproteins involvesthe extracellular matrix; proteoglycans in particular have beenhypothesized to play an important role (Hurt-Camejo, E. et al. 1997.Arterioscler Thromb Vasc Biol. 17:1011-1017; Williams, K. J., and I.Tabas. 1995. Arterioscier. Thromb. Vasc Biol. 15:551-561; Camejo, G. etal. 1988. Arteriosclerosis. 8:368-377; and Hurt, E., and G. Camejo.1987. Atherosclerosis. 67:115-126). First, purified arterialproteoglycans, especially those from lesion-prone sites (Cardoso, L. E.,and P. A. Mourao. 1994. Arterioscler. Thromb. 14:115-124; and Ismail, N.et al. 1994. Atherosclerosis. 105:79-87), bind atherogenic lipoproteinsin vitro, particularly LDL from patients with coronary artery disease(Linden, T. et al. 1989. Eur. J. Clin. Invest. 19:38-44). LDL binds withhigh affinity to dermatan sulfate and chondroitin sulfate proteoglycansproduced by proliferating smooth muscle cells (Camejo, G. et al. 1993.J. Biol Chem. 268:14131-1437). Second, proteoglycans are a majorcomponent of the artery wall extracellular matrix and are available toparticipate in the interactions of lipoproteins in the earliest stagesof atherogenesis. Third, retained apo-B immunologically co-localizeswith proteoglycans in early and developed lesions (Walton, K., and N.Williamson. 1968. J. Atheroscler. Res. 8:599-624; Hoff, H., and G. Bond.1983. Artery. 12:104-116; Hoff, H. F., and W. D. Wagner. 1986.Atherosclerosis. 61:231-236; Nievelstein-Post, P. et al. 1994.Arterioscler. Thromb. 14:1151-1161; and Galis, Z. et al. 1993. Am J.Pathol. 142:1432-1438). The observation that the arterial wall contentof these proteoglycans increases during atherosclerosis and correlateswith an increased accumulation of aortic cholesterol also supports thepotential importance of the interaction between LDL and proteoglycans(Hoff, H. F., and W. D. Wagner. 1986. Atherosclerosis. 61:231-236;Merrilees, M. et al. 1990. Arteriosclerosis. 81:245-254).

Proteoglycans contain long carbohydrate side-chains ofglycosaminoglycans, which are covalently attached to a core protein by aglycosidic linkage. The glycosaminoglycans consist of repeatingdisaccharide units, all bearing negatively charged groups, usuallysulfate or carbohydrate groups. In vitro, LDL bind with high affinity tomany proteoglycans found in the artery wall, including dermatan sulfateproteoglycans (e.g., biglycan) and chondroitin sulfate proteoglycans(e.g., versican), which are produced by smooth muscle cells in responseto PDGF or TGFβ (Schonherr, E. et al. 1991. J. Biol. Chem.266:17640-17647; and Schönherr, E. et al. 1993. Arterioscler. Thromb.13:1026-1036). The interaction between LDL and proteoglycans have beenhypothesized to involve clusters of basic amino acids in apo-B100, theprotein moiety of LDL, that interact with the negatively chargedglycosaminoglycan proteoglycans (Mahley, R. et al. 1979. Biochem.Biophys. Acta. 575:81-91; Camejo, G. et al. 1988. Arteriosclerosis.8:368-377; Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem.262:11097-11103; and Hirose, N. et al. 1987. Biochemistry. 26:5505-5512)or by bridging molecules such as apo-E or lipoprotein lipase (Williams,K. J., and I. Tabas. 1995. Arterioscier. Thromb. Vasc. Biol.15:551-561).

Isolation of large fragments of apo-B100 from different regionscharacterized by concentrations of positive clusters indicated that upto eight specific regions in apo-B100 bind proteoglycans (Camejo, G. etal. 1988. Arteriosclerosis. 8:368-377; Weisgraber, K., and S. Rall, Jr.1987. J. Biol. Chem. 262:11097-11103; and Hirose, N. et al. 1987.Biochemistry. 26:5505-5512). Weisgraber, K., and S. Rall, Jr. 1987. J.Biol. Chem. 262:11097-11103 identified two fragments, residues 3134-3209and 3356-3489, that bind to heparin with the highest affinity. RecentlyCamejo and coworkers confirmed this finding and proposed that residues3147-3157 and 3359-3367 may act cooperatively in the association withproteoglycans (Hurt-Camejo, E. et al. 1997. Arterioscler Thromb VascBiol. 17:1011-1017; and Olsson, U. et al. 1997. Arterioscler. Throm.Vasc. Biol. 17:149-155). However, because these studies were carried outwith delipidated apo-B fragments in the presence of urea or with shortsynthetic apo-B peptides, it is not clear which of the binding sites arefunctionally expressed on the surface of LDL particles. Some or many ofthese postulated glycosaminoglycan-binding sites may not be functionalwhen apo-B is associated with LDL. For example, apo-E has twoheparin-binding sites, but only one binds to heparin when apo-E iscompleted with phospholipid (Weisgraber, K. et al. 1986. J. Biol Chen261:2068-2076). This heparin-binding site coincides with the LDLreceptor-binding site of apo-E.

Although eight potential glycosaminoglycan-binding sites have beenidentified in apo-B100 (Camejo, G. et al. 1988. Arteriosclerosis.8:368-377; Weisgraber, K., and S. Rall, Jr. 1987. J. Biol. Chem.262:11097-11103; and Hirose, N. et al. 1987. Biochemistry.26:5505-5512), it was not known which of them participate in thephysiological binding of LDL to proteoglycans. Previously, we havedemonstrated, in conjunction with others, that Site B (residues3359-3369) is the LDL receptor-binding site, and in the study whichgenerated the present invention we found that it is also the primarybinding site to proteoglycans.

Modification of LDL potentially exposes the other proteoglycan-bindingsites. Paananen and Kovanen (Paananen, K., and P. T. Kovanene. 1994. J.Biol. Chem. 269:2023-2031) noted that proteolysis of apo-B100strengthened the binding of LDL to proteoglycans, suggesting theexposure of buried heparin binding sites. Likewise, when LDL are fusedby sphingomyelinase treatment, the modified lipoproteins bind moreavidly to proteoglycans. The finding that multiple heparin moleculesbind to LDL (Cardin, A. et al. 1987. Biochemistry. 26:5513-5518) mayalso be explained by a cooperative effect of heparin binding to one sitethat triggers a conformational change in apo-B100 that enables othersites to participate in the interaction. Thus, the initial interactionwith proteoglycans may induce structural alterations of the LDL thatexpose heparin/proteoglycan-binding sites that may contribute to theintramural retention of LDL after the initial interaction with theprimary binding site.

The interaction between LDL and the LDL receptor plays a major role indetermining plasma cholesterol levels in humans and other mammalianspecies (Goldstein, J. et al. 1985. Annu. Rev. Cell Biol. 1:1-39).Apo-B100 is the major protein component of LDL and is responsible forthe binding of these lipoproteins to the LDL receptor (Innerarity, T. etal. 1990. J. Lipid Res. 31:1337-1349). The relevance of this catabolicpathway is best illustrated by the genetic disorders familialhypercholesterolemia (FH) and familial defective apo-B100 (FDB), inwhich high levels of LDL accumulate in the circulation because mutationsin the LDL receptor (FH) or in the ligand (FDB) disrupt the binding ofLDL to its receptor (Innerarity, T. et al. 1990. J. Lipid Res.31:1337-1349). Many different mutations of the LDL receptor cause FH(Hobbs, H. et al. 1992. Hum. Mutat. 1:445-466), but FDB is associatedwith a single site mutation, the substitution of glutamine (Innerarity,T. et al. 1987. Proc. Natl. Acad. Sci. USA. 84:6919-6923) or, in a fewcases, tryptophan (Gaffney, D. et al. 1995. Arterioscler. Thromb. Vasc.Biol. 15:1025-1029) for the normally occurring arginine at residue 3500of apo-B100. With the exception of an arginine-3531 to cysteine mutation(Pullinger, C. et al. 1995. J. Clin. Invest. 95:1225-1234), which isassociated with a minor decrease in LDL receptor binding, extensivesearches have not found any other mutations of apo-B100 that causedefective receptor binding of LDL (Pullinger, C. et al. 1995. J. Clin.Invest. 95:1225-1234). The FDB mutation occurs at an estimated frequencyof 1/500 in the normal population and is therefore one of the mostcommon known single-gene defects causing an inherited abnormality(Innerarity, T. et al. 1990. J. Lipid Res. 31:1337-1349).

Much attention has focused on understanding the molecular interactionbetween apo-B100 and the LDL receptor. The structural and functionaldomains of the LDL receptor have been defined in detail (Hobbs, H. etal. 1992. Hum. Mutat. 1:445-466), but much less is understood about thereceptor-binding domain of apo-B100, because of its large size andinsolubility in aqueous buffer. Furthermore, both the lipid compositionand the conformation of apo-B100 appear to be crucial to its function asan effective ligand for the LDL receptor, since apo-B100 binds to theLDL receptor only after the conversion of large VLDL to smaller LDL(Goldstein, J. et al. 1985. Annu. Rev. Cell Biol. 1:1-39).

Selective chemical modification of the apo-B100 of LDL demonstrated thatthe basic amino acids arginine and lysine were important in theinteraction of LDL with its receptor (Mahley, R. et al. 1977. J. Biol.Chem. 252:7279-7287; and Weisgraber, K. et al. 1978. J. Biol. Chem.253:9053-9062). Once apo-B100 was sequenced, several regions enriched inarginine and lysine residues became candidates for receptor binding,including Site A (residues 3147-3157) and Site B (residues 3359-3367)(Knott, T. et al. 1985. Science. 230:3743).

While it had been hypothesized that LDL-proteoglycan binding waspossibly important to the formation of atherosclerotic lesions throughthe retention of lipoproteins in the subendothelium, this hypothesis hasnot been empirically demonstrated in the art. Moreover, there have beensix obstacles which have prevented other researchers from demonstratingthe mechanism by which atherogenesis occurs and using this informationto combat atherosclerosis. First, there have been eight potential sitesidentified in the apo-B100 protein, any one or several of which couldhave been responsible for proteoglycans trapping LDL in thesubendothelium. Second, it has been unknown which potential sites in theapo-B100 are exposed to the surface of the LDL particles and which areburied within the lipid core. Third, there has been evidence that someof the potential proteoglycan binding sites on apo-B100 may workcooperatively, creating the possibility that blocking proteoglycanbinding at any single site might not have proven both necessary andsufficient to eliminate LDL retention in the subendothelium. Fourth, themodification of LDL has been shown in some cases to expose newproteoglycan binding sites to the surface. Fifth, any disruption to LDLproteoglycan binding had the potential to disrupt LDL receptor binding,which would serve to disrupt the natural clearance of LDL from blood,raise serum cholesterol levels, and potentially result in a conditionsimilar to familial hypercholesterolemia. Sixth, it has not beenpossible to use site-directed mutagenesis and express the entire mutatedapo-B100 proteins as LDL in order to define the proteoglycan-bindingsites on LDL.

SUMMARY OF THE INVENTION

We have discovered that the amino acids of Site B in the apo-B100protein are responsible for conferring proteoglycan binding activity onLDL. Recombinant LDL in which lysine₃₃₆₃ in apo-B100 was changed toglutamic acid has severely defective proteoglycan binding activity butnormal LDL receptor-binding activity. Thus, the proteoglycan-binding andthe receptor-binding activities in LDL can be separated by theintroduction of a single point mutations into the apo-B100 protein,indicating that pharmaceutical strategies for disruptingLDL-proteoglycan binding need not inhibit LDL receptor binding.

Moreover, we have demonstrated for the first time in vivo thatLDL-proteoglycan binding is necessary to the formation ofatherosclerotic lesions and the onset of atherosclerosis. Transgenicmice expressing the mutant RK3359-3369SA apo-B100 LDL, which isdefective for proteoglycan binding, was found to have strikingly lessatherosclerosis than mice expressing the wild-type recombinant LDL, whenboth were fed a high cholesterol diet. These results demonstrate thatdisruption of LDL-proteoglycan binding at Site B in the apo-B100 proteinis a credible target for pharmaceutical intervention for the reductionand elimination of atherosclerosis.

The present invention relates to the prevention of atherosclerosisthrough the modulation of LDL-proteoglycan binding at Site B (aminoacids 3359-3369) of the apo-B100 protein in LDL. The inventionencompasses apo-B100 proteins with mutations in Site B and which exhibitreduced binding to proteoglycans, fragments of these proteins containingSite B, and LDL particles comprising such mutants. The inventionincludes purified apo-B100 proteins comprising a mutation in Site Bwhich results in reduced LDL-proteoglycan binding activity whilemaintaining LDL/LDL receptor binding (proteoglycan⁻receptor⁺ mutant),including, for example, the K3363E mutation. The inventions alsoincludes polypeptide fragments of these proteins which comprise theamino acid sequence of Site B in the apo-B100 protein of the invention,wherein said Site B is flanked on at least one side by a contiguoussequence of amino acids which is directly adjacent to Site B in thewild-type human apo-B100 sequence. The invention encompasses LDLparticles and other lipoproteins which comprise an apo-B100 protein orprotein fragment of the invention.

Accordingly, in certain embodiments, the invention provides mutantapo-B100 proteins and mutant apo-B100 polypeptide fragments, as well asLDL particles and other lipoproteins comprising a mutant apo-B100protein or polypeptide fragment, which comprise a mutant Site B selectedfrom one of the following Site B sequences:

-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Glu₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆ys₃₃₆₇    (SEQ ID NO:1);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Asp₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:2);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Ala₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:3);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Thr₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:4);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Ser₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:5);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Gln₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:6);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Glu₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:7);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Asp₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:8);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Glu₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:9);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Asp₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:10);-   Thr₃₃₅₈-Glu₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:11);    and-   Thr₃₃₅₈-Asp₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:12);    as well as Site B sequences with deletions, such as:-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg3362 - - -    Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇; (SEQ ID NO:13);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁ - - -    Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇(SEQ ID NO:14);    and-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃ - - -    Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇(SEQ ID NO:15);    and Site B sequences which include insertions, such as:-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Glu-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:16);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Glu-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:17);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Asp-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:18);-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Asp-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:19);

The invention also includes antibodies which bind to antigenicdeterminants comprising Site B of the mutant apo-B100 proteins of theinvention, including antibody compositions which bind to an antigenicdeterminant in an apo-B100 protein or protein fragment of the invention,wherein said antigenic determinant is not present in the wild-type humanapo-B100 protein.

The invention also encompasses polynucleotides encoding the mutantapo-B100 proteins of the invention, targeting vectors and methods forcreating mutant apo-B100 genes of the invention. The invention includespolynucleotides which encode an apo-B100 protein or protein fragment ofthe invention, as well as cells comprising a polynucleotide of theinvention or expressing an apo-B100 protein or protein fragment of theinvention. The invention also includes non-human animals and mammalswhich comprise a polynucleotide of the invention or express an LDL,apo-B100 protein, or protein fragment of the invention.

The invention encompasses methods for preventing or reducing theseverity of atherosclerosis in an animal or mammal, comprising the stepof expressing a polynucleotide, LDL, apo-B100 protein, or proteinfragment of the invention. Normally, a polynucleotide encoding anapo-B100 protein or protein fragment of the invention is transduced intoa cell. The cell may be transduced ex vivo, then transferred into theanimal or mammal, or the cell may be transduced in situ.

The present invention further encompasses methods of screening for andidentifying inhibitors of LDL-proteoglycan binding, including drugscreening assays based on simple LDL-proteoglycan binding, highthrough-put drug screening assays based on LDL-proteoglycan binding, twostep LDL/proteoglycan and LDL/LDL-receptor binding assays, and intransgenic animals which express recombinant LDL.

The present invention encompasses methods for identifying inhibitors ofLDL-proteoglycan binding, comprising the steps of:

(a) incubating a mixture comprising (i) proteoglycan, (ii) LDL, and(iii) a candidate compound, under conditions wherein LDL binds toproteoglycan to form an LDL-proteoglycan complex in the absence of saidcandidate compound;

(b) determining any difference between the amount of LDL-proteoglycancomplex present in:

-   -   (i) the mixture prepared in step (a), and    -   (ii) a control mixture comprising said proteoglycan and said LDL        in the absence of said candidate compound; and optionally

(c) correlating any difference determined in step (b) with saidcandidate compound's ability to affect LDL-proteoglycan binding.

The present invention also encompasses identifying compounds whichaffect LDL-proteoglycan binding, which do not substantially affect LDLreceptor binding, which further comprising the steps of:

(d) incubating a mixture comprising (i) LDL receptor, (ii) LDL, and(iii) a candidate compound that affects LDL-proteoglycan bindingidentified in step (c), under conditions wherein LDL binds to LDLreceptor to form an LDL-LDL receptor complex in the absence of saidinhibitor of LDL-proteoglycan binding;

(e) determining any difference between the amount of LDL-LDL receptorcomplex present in:

-   -   (i) the mixture prepared in step (d), and    -   (ii) a control mixture comprising said LDL receptor and said LDL        in the absence of said inhibitor of LDL-proteoglycan binding;        and optionally

(f) correlating any difference determined in step (e) with the LDL-LDLreceptor binding activity of said candidate compound that affectsLDL-proteoglycan binding.

In accordance with the instant invention, either the LDL or theproteoglycan of step (a) may be adhered to a solid support.Additionally, where the LDL is adhered to a solid support, theproteoglycan may be labeled, or where the proteoglycan is adhered to asolid support, the LDL may be labeled.

The invention further encompasses methods for identifying compoundswhich modulate atherosclerosis and/LDL-proteoglycan binding in vivo,comprising the steps of:

(a) administering a candidate compound to a transgenic non-human animalwhich expresses a human apo-B gene, under conditions wherein measurableatherosclerotic lesions form in the arteries of said animal in theabsence of said candidate compound;

(b) determining any difference between the extent of atherosclerosispresent in:

-   -   (i) the animal of step (a), and    -   (ii) a control transgenic non-human animal in the absence of        said candidate compound; and optionally

(c) correlating any difference determined in step (b) with the saidcandidate compound's ability to modulate atherosclerosis in vivo.

The present invention further encompasses the compounds identified bythe screening methods of the invention, including the compounds whichaffect, modulate, stimulate or inhibit of LDL-proteoglycan bindingidentified by the methods for identifying compounds that affectLDL-proteoglycan binding, as well as the compounds that affect,modulate, stimulate, or inhibit LDL-proteoglycan binding, which do notsubstantially affect LDL receptor binding identified by the methods foridentifying inhibitors of LDL-proteoglycan binding, which do noteliminate LDL receptor binding, and the compounds which modulate,stimulate, or inhibit atherosclerosis in vivo identified by the methodsfor identifying compounds which modulate atherosclerosis in vivo. Inaddition the invention encompasses methods of inhibiting atherosclerosisin a human comprising administering to the human an agent that inhibitsLDL-proteoglycan binding, or any of the other compounds identified bythe methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a half tone reproduction of a Coomassie staining and westernanalysis of recombinant LDL. Recombinant LDL (d=1.02-1.05 g/ml) fromfour lines of human apo-B transgenic mice were isolated by sequentialultracentrifugation and subjected to immunoaffinity chromatography toremove endogenous apo-B and apo-E. Five micrograms of apo-B100 fromhuman plasma LDL (lane 1) or recombinant LDL: control LDL (lane 2),R3500Q LDL (lane 3), RK3359-3369SA LDL (lane 4), and K3363E LDL (lane 5)were analyzed by SDS-PAGE with 3-15% gels (FIG. 1A). One microgram eachof unpurified LDL (lane 1) and control LDL (lane 2), R3500Q LDL (lane3), RK3359-3369SA LDL (lane 4), and K3363E LDL (lane 5) were analyzed bywestern blots with monoclonal antibody 1 D 1 against human apo-B (FIG.1B) and polyclonal antibodies against mouse apo-B (FIG. 1C) or mouseapo-E (FIG. 1D).

FIG. 2 is a graph demonstrating a competitive binding assay ofrecombinant LDL. The abilities of recombinant control LDL (closedtriangle), R3500Q LDL (closed square), RK3359-3369SA LDL (open diamond),and K3363E LDL (open circle) to compete with ¹²⁵I-labeled human plasmaLDL (2 μg/ml) for binding to LDL receptors on normal human fibroblastswere determined. The recombinant lipoproteins were isolated from 15mice, and endogenous apo-E and apo-B were removed. Competitor LDL wereadded at the indicated concentrations to normal human fibroblasts, andthe amount of ¹²⁵I-LDL bound to the fibroblasts was measured after a 3-hincubation. The results represent the average of data from threeindependent experiments performed with freshly isolated LDL for eachexperiment human plasma LDL (closed circle) was included as a control.

FIG. 3 is a graph of a gel-shift analysis of mouse-derived recombinantLDL with (³⁵S)biglycan and (³⁵S)versican. The abilities of recombinantcontrol LDL (closed triangle), R3500Q LDL (closed square), RK3359-3369SALDL (open diamond), and K3363E LDL (open circle) to interact with(³⁵S)biglycan (FIG. 3A) and (³⁵S)versican (FIG. 3B) were determined. Therecombinant lipoproteins were isolated from 15 mice, and endogenousapo-E and apo-B were removed by immunoaffinity chromatography. Theresults represent the average data from three independent experimentsperformed with freshly isolated LDL for each experiment. Human plasmaLDL (closed circle) was included as a control.

FIG. 4 is a graph of a gel-shift analysis of mouse-derived recombinantLDL with (³⁵S)versican and (³⁵S)biglycan after selective modification.The abilities of recombinant control LDL (closed circle) andRK3359-3369SA LDL (closed diamond), cyclohexanedione-modified controlLDL (closed triangle, point down) and RK3359-3369SA LDL(closed triangle,point up), and acetylated control LDL (open circle) and RK3359-3369SALDL (closed square) to interact with (³⁵S)versican (FIG. 4A) or(³⁵S)biglycan (FIG. 4B) were determined. The recombinant lipoproteinswere isolated from 15 mice, and endogenous apo-E and apo-B were removedby immunoaffinity chromatography. The isolated recombinant LDL weretreated with (FIG. 4A) acetic anhydride or (FIG. 4B) cyclohexanedione toselectively modify all arginines or lysines, respectively, in apo-B100.

FIG. 5 is a graph demonstrating the correlation between the percentageof total aortic surface area covered by lesions and the plasmacholesterol levels of transgenic mice expressing either normal humanrecombinant LDL (open circle) or defective-proteoglycan-binding LDL(closed circle) after the mice had been fed a high-fat, high-cholesteroldiet for 17 weeks.

FIG. 6 is a half-tone reproduction of photographs of Sudan IV-stainedaorta from a mouse expressing human wild-type recombinant LDL (top),proteoglycan-binding-defective LDL (center), and endogenous LDL(bottom). The wild-type recombinant LDL mouse and the RK3359-3369SA LDLmouse had plasma cholesterol levels of 678 and 616 mg/dl, respectively.

DISCLOSURE OF THE INVENTION

The practice of the present invention encompasses conventionaltechniques of chemistry, immunology, molecular biology, biochemistry,protein chemistry, and recombinant DNA technology, which are within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Oligonucleotide Synthesis (M. Gait ed. 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, eds., 1984); Sambrook, Fritsch &Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989);PCR Technology (H. A. Erlich ed., Stockton Press); R. Scope, ProteinPurification Principles and Practice (Springer-Verlag); and the seriesMethods in Enzymology (S. Colowick and N. Kaplan eds., Academic Press,Inc.).

Definitions

The terms “LDL” or “low density lipoprotein” refers to a particle with adiameter of approximately 22 nm and a mass of about three million Daltonfound in plasma. LDL is comprised of a highly hydrophobic core ofapproximately 1500 cholesteryl ester molecules surrounded by a shell ofphospholipids, unesterified cholesterol, and a single apo-B100 protein.LDL is often differentiated and separated from other plasma lipoproteinsby its density of 1.019 to 1.063 g/ml through ultracentrifugation asdescribed in Example 4. As used herein the term “LDL” embraceslipoprotein particles comprising a mutant apo-B100 protein, as well aslipids which do not naturally occur in LDL and labels, all of which maychange the physical properties listed above. In all cases an LDLparticle contains only one apolipoprotein, a apo-B100 protein orfragment thereof, and contains a lipid core which is predominantlycholesteryl ester.

The “apo-B100 protein” resides in the outer shell of very low densitylipoproteins (VLDL), intermediate density lipoproteins (IDL), and lowdensity lipoproteins (LDL). The complete sequence and identification ofstructural domains of human apo-B100 protein is found in Knott, T. et al1986. Nature 323:734-738.

Apo-B100 is the component of LDL which binds specifically to the “LDLreceptor” on the plasma membrane of non-hepatic cells. The LDL receptorsare localized in specialized regions called coated pits, where the“LDL/LDL receptor complex” is internalized through endocytosis,delivering cholesterol to the cell. As used herein the “LDL receptor”and any resulting “LDL/LDL receptor complex” need not contain anyportion of the native LDL receptor which is not needed to achieveLDL-binding. Thus, only a sufficient portion of the 292 amino-terminalamino acid LDL-binding domain of the native LDL receptor and any otherdomains which are necessary to confer binding to LDL need be present inan “LDL receptor.”

As used herein, the term “purified apo-B100 protein” refers to anapo-B100 protein isolated from a lipoprotein, including wild-typeapo-B100, mutant apo-B100 and protein fragments thereof, which isessentially free, i.e., contains less than about 50%, preferably lessthan about 30%, and more preferably less than about 10%, even morepreferably less than about 5%, and still more preferably less than about1% of the lipids with which an apo-B100 protein is normally associatedin a lipoprotein.

As used herein the term “Site B” refers to amino acids from about 3359to about 3369 of the human apo-B100 protein.

The terms “human recombinant LDL” and “recombinant LDL” are usedinterchangeably herein to refer to LDL populations comprising LDLparticles derived from a non-human animal which contains a humanapo-B100 protein. The human apo-B100 proteins contained within arecombinant LDL may be wild-type apo-B100 protein. Without expressmention, the human apo-B100 protein of a recombinant LDL may also have aleucine in place of the glutamine residue at position 2153, whichabolishes the formation of apo-B48, resulting in a higher yield ofrecombinant apo-B100 LDL. In addition the human apo-B100 proteins ofrecombinant LDL may have other mutations which are expressly noted intheir name (e.g., K3363E LDL). In addition the term “recombinant LDL”embraces any LDL reagent which comprises at least a fragment of arecombinant apo-B100 protein and maintains the LDL-proteoglycan bindingactivity of at least 60% of wild-type levels, preferably at least 70%,more preferably at least 80%, still more preferably 90%, most preferablyat essentially 100% of wild-type LDL-proteoglycan binding activity. Thephrase “recombinant control LDL” is used herein to refer to LDL,containing a human apo-B100 protein in which the glutamine at amino acidposition 2153 has been replaced with a leucine.

As used herein the term “R3500Q” refers to a human apo-B100 protein inwhich the naturally-occurring arginine at residue 3500 of the humanapo-B100 protein has been replaced with a glutamine residue. The term isalso used to refer to genes and plasmids which encode the R3500Q mutantapo-B100 protein, as well as recombinant LDL which comprises the mutantprotein and transgenic mice or other non-human animals which express theR3500Q recombinant LDL.

As used herein the term “K3363E” refers to a human apo-B100 proteinwherein the naturally-occurring lysine at residue 3363 of the humanapo-B100 protein has been replaced with a glutamic acid residue. Theterm is also used to refer to genes and plasmids which encode the K3363Emutant apo-B100 protein, as well as recombinant LDL which comprises themutant protein and transgenic mice or other non-human animals whichexpress the K3363E recombinant LDL.

As used herein the term “RK3359-3369SA” refers to a human apo-B100protein in which the basic amino acids in Site B (residues 3359-3369)were converted to neutral amino acids with all of the arginine residuesbeing converted to serines and the lysine residues being converted toalanines. The term is also used to refer to genes and plasmids whichencode the RK3359-3369SA mutant apo-B100 protein, as well as recombinantLDL which comprises the mutant protein and transgenic mice or othernon-human animals which express the RK3359-3369SA recombinant LDL.

As used herein the term “proteoglycan⁻receptor⁺” is used to refer mutantapo-B100 proteins, fragments thereof as well as LDL comprising thesepolypeptides and transgenic non-human animal strains which express theseproteins. A proteoglycan⁻receptor⁺ apo-B100 protein when present in anLDL particle reduces proteoglycan binding of that LDL particle by atleast 50%, preferably by at least 60%, more preferably by at least 70%,still more preferably by at least 80%, even more preferably by at least90%, most preferably by 95% or greater. Proteoglycan binding may beassayed by any method known in the art. See, for example, the methoddescribed in Example 8. In addition a proteoglycan⁻receptor⁺ apo-B100protein when present in an LDL particle confers LDL receptor bindingactivity to that LDL particle of at least 60% of wild-type levels,preferably at least 70%, more preferably at least 80%, still morepreferably 90%, most preferably at essentially 100% of wild-type LDLreceptor binding activity.

The amino acid sequence of the wild-type human apo-B100 protein fromamino acid 3358 to 3367 is as follows:

-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:20).

The proteoglycan⁻receptor⁺ mutant apo-B100 proteins of the inventioninvolve substitutions or deletions at the following four amino acidpositions: Lys₃₃₆₃, Arg₃₃₆₂, Arg₃₃₆₄, and Arg₃₃₅₉. Aproteoglycan⁻receptor⁺ mutant of the invention can be constructed by thesubstitution or deletion of any single one of these amino acids, anycombination or them or all four of the amino acids in positions 3363,3362, 3364, and 3359. Preferably two or fewer of these amino acids aresubstituted or deleted, more preferably only a single amino acid issubstituted or deleted. When only a single amino acid is chosen to besubstituted or deleted, preferably the amino acid which is substitutedor deleted is one of positions 3363, 3362, and 3364, more preferablyposition 3363. While any amino acid can be used in a substitution,preferably the new amino acid is chosen from the group consisting ofGly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Cys, Met, Asn, Gln, Asp, andGlu, more preferably the new amino acid is either Asp or Glu.

In addition to deletions and substitution, a proteoglycan⁻receptor⁺apo-B100 proteins can be formed by additions to the amino acid sequence.Additions are usually only a single amino acid, and can be made to oneor more of the following locations: between 3358 and 3359, between 3359and 3360, between 3361 and 3362, between 3362 and 3363, between 3363 and3364, and between 3364 and 3365. Preferably additional amino acids areadded to two or fewer of these sites, more preferably an addition ismade to only one of these sites. When only a single position is chosenfor an addition preferably that site is either between 3362 and 3363, orbetween 3363 and 3364. While any amino acid may be added for theseadditions, preferably amino acids for addition are chosen from thefollowing list Gly, Ala, Val, Leu, Ile, Phe, Tyr, Tip, Cys, Met, Asn,Gln, Asp, and Glu, more preferably the new amino acid is Ala, Ser, Thr,Gln, Asp or Glu, even more preferably the new amino acid is Asp or Glu.It should be noted that combinations of the additions, deletions andsubstitutions described can be employed to construct aproteoglycan⁻receptor⁺ apo-B100 protein. These changes to the nativeprotein may be achieved by any method known in the art, includingchemical synthesis or modification. However, expression of recombinantapo-B100 gene made by site-directed mutagenesis as demonstrated, forexample, in Examples 1-3 is preferred.

The following are the amino acid sequences from position 3358 toposition 3367 for a list of preferred proteoglycan⁻receptor⁺ apo-B100protein mutants:

-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Glu₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:1)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Asp₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:2)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Ala₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:3)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Thr₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:4)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Ser₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:5)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Gln₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:6)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Glu₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:7)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Asp₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:8)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Glu₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:9)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Asp₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:10)-   Thr₃₃₅₈-Glu₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:11)-   Thr₃₃₅₈-Asp₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:12)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂ - - -    Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇ (SEQ ID NO:13)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁ - - -    Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇ (SEQ ID NO:14)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃ - - -    Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇ (SEQ ID NO:15)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Glu-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:16)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Glu-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:17)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Asp-Lys₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:18)-   Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Lys₃₃₆₃-Asp-Arg3364-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇    (SEQ ID NO:19), wherein the repeated dashed lines represent    deletions.

The term “proteoglycan” refers to a class of compounds with a highrelative molecular mass which comprise carbohydrate and protein, and arefound in animal structural tissues, e.g. the ground substance ofcartilage and bone. The ground substance and gel fluids of these tissuesowe their viscosity and elasticity to the presence of proteoglycans.Each proteoglycan contains 40 to 80 mucopolysaccharide chains(glucosaminoglycans) usually bound to the protein via o-glycosidiclinkages to serine or threonine. In contrast to the glycoproteins, theprosthetic group of proteoglycans has a relative molecular mass of20,000 to 30,000, consisting of many (approximately 100-1000)unbranched, regularly repeating disaccharide units. The disaccharidesare composed of a derivative of an amino sugar, either glucosamine orgalactosamine. At least one of the sugars in the disaccharide has anegatively charged carboxylate or sulfate group. Hyaluronate,chondroitin sulfate, keratin sulfate, heparin sulfate, and heparin arethe most common glucosaminoglycans. Heterogeneity of proteoglycans isdue to differences in polypeptide chain length, and to the number anddistribution of the attached polysaccharide chains.

Microheterogenicity also exists, due to small differences in the chainlengths of the polysaccharide chains, and the distribution of sulfateresidues for a particular type of proteoglycan.

As used herein, the term “atherosclerosis” refers to a disease statecharacterized by irregularly distributed deposits of lipid andlipoprotein in the intima of large and medium-sized arteries oftencovered with a fibrous cap and calcification. The terms “atheroscleroticlesions” and “atherosclerotic plaques” are used interchangeably hereinto refer to these deposits.

As used herein, the term “non-human animal” refers to any non-humanvertebrate, birds and more usually mammals, preferably primates, farmanimals such as swine, goats, sheep, donkeys, and horses, rabbits orrodents, more preferably rats or mice. As used herein, the term “animal”is used to refer to any vertebrate, preferable a mammal. Both the terms“animal” and “mammal” expressly embrace human subjects unless precededwith the term “non-human”.

As used interchangeably herein, the term “oligonucleotides”, and“polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of morethan one nucleotide in either single chain or duplex form. The term“nucleotide” as used herein as an adjective to describe moleculescomprising RNA, DNA, or RNA/DNA hybrid sequences of any length insingle-stranded or duplex form. The term “nucleotide” is also usedherein as a noun to refer to individual nucleotides or varieties ofnucleotides, meaning a molecule, or individual unit in a larger nucleicacid molecule, comprising a purine or pyrimidine, a ribose ordeoxyribose sugar moiety, and a phosphate group, or phosphodiesterlinkage in the case of nucleotides within an oligonucleotide orpolynucleotide. Although the term “nucleotide” is also used herein toencompass “modified nucleotides” which comprise at least onemodifications (a) an alternative linking group, (b) an analogous form ofpurine, (c) an analogous form of pyrimidine, or (d) an analogous sugar,for examples of analogous linking groups, purine, pyrimidines, andsugars see for example PCT publication No. WO 95/04064. However, thepolynucleotides of the invention are preferably comprised of greaterthan 50% conventional deoxyribose nucleotides, and most preferablygreater than 90% conventional deoxyribose nucleotides. Thepolynucleotide sequences of the invention may be prepared by any knownmethod, including synthetic, recombinant, ex vivo generation, or acombination thereof, as well as utilizing any purification methods knownin the art.

The term “purified” is used herein to describe a polynucleotide orpolynucleotide vector of the invention which has been separated fromother compounds including, but not limited to other nucleic acids, andproteins (such as the enzymes used in the synthesis of thepolynucleotide), or the separation of covalently closed polynucleotidesfrom linear polynucleotides. A polynucleotide is substantially pure whenat least about 60 to 75% of a sample exhibits a single polynucleotidesequence and conformation (linear versus covalently close). Asubstantially pure polynucleotide typically comprises about 60 to 90%weight/weight of a nucleic acid sample, more usually about 95%, andpreferably is over about 99% pure. Polynucleotide purity or homogeneitymay be indicated by a number of means well known in the art, such asagarose or polyacrylamide gel electrophoresis of a sample, followed byvisualizing a single polynucleotide band upon staining the gel. Forcertain purposes higher resolution can be provided by using HPLC orother means well known in the art.

As used herein, the term “antibody” means an immunoglobulin molecule ora fragment of an immunoglobulin molecule having the ability tospecifically bind to a particular antigen. Antibodies are well known tothose of ordinary skill in the science of immunology. As used herein,the term “antibody” means not only intact antibody molecules but alsofragments of antibody molecules retaining antigen binding ability. Suchfragments are also well known in the art and are regularly employed bothin vitro and in vivo. In particular, as used herein, the term “antibody”means not only intact immunoglobulin molecules of any isotype (IgA, IgG,IgE, IgD, IgM) but also the well-known active (i.e., antigen-binding)fragments F(ab′)₂, Fab, Fv, scFv, Fd, V_(H) and V_(L). For antibodyfragments, see, for example “Immunochemistry in Practice” (Johnstone andThorpe, eds., 1996; Blackwell Science), p. 69. The term “antibody”further includes single chain antibodies, CDR-grafted antibodies,chimeric antibodies, humanized antibodies, and a Fab expression library.The term also includes fusion polypeptides comprising an antibody of theinvention and another polypeptide or a portion of a polypeptide (a“fusion partner”). Examples of fusion partners include biologicalresponse modifiers, lymphokines, cytokines, and cell surface antigens.

As used herein, an “antigenic determinant” is the portion of an antigenmolecule, in this case a mutant apo-B100 protein, that determines thespecificity of the antigen-antibody reaction. An “epitope” refers to anantigenic determinant of a polypeptide. An epitope can comprise as fewas 3 amino acids in a spatial conformation which is unique to theepitope. Generally an epitope consists of at least 6 such amino acids,and more usually at least 8-10 such amino acids. Methods for determiningthe amino acids which make up an epitope include x-ray crystallography,2-dimensional nuclear magnetic resonance, and epitope mapping e.g. thePepscan method described by H. Mario Geysen et al. 1984. Proc. Natl.Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No. WO 84/03564; and PCTPublication No. WO 84/03506.

Methods for Identifying Compounds that Affect of LDL-ProteoglycanBinding

The present invention provides new assay methods for detecting, andpreferably quantifying, one or more compounds that affectLDL-proteoglycan binding of interest which are present in a library ofcandidate compounds. Identifying compounds which inhibitLDL-proteoglycan activity is the preferred use of this assay, but it canequally be used to identify compounds which result in an increase inLDL-proteoglycan activity. The terms “assay” and “assay method,” as usedherein, pertain to a method of detecting the presence of (e.g.,qualitative assay), and preferably quantifying (e.g., quantitativeassays), the modulation of LDL-proteoglycan binding.

Assays of the present invention generally involve contacting thecandidate compound of interest with a pre-determined non-limiting amountof both an LDL reagent and a proteoglycan reagent, measuring theLDL-proteoglycan binding which results, and correlating the measuredLDL-proteoglycan binding with the candidate compound's ability to affector modulate LDL-proteoglycan binding. In a qualitative assay, simplydetermining whether the measured LDL-proteoglycan binding is above orbelow a threshold value (established, for example, using recombinant LDLsamples with known LDL-proteoglycan binding properties) may besufficient to establish the assay result. Typically, when the effect isan inhibition the relationship is determined from standard samplescontaining known amounts of a competitive inhibitor of LDL-proteoglycanbinding. Such competitive inhibitors can include, depending on the assaya non-labeled LDL or proteoglycan which has normal binding activity.Thus, unless otherwise required, the term “measuring” can refer toeither qualitative or quantitative determination.

The terms “agent” or “candidate compound” as used interchangeablyherein, pertain to a substance which is to be measured for a possibleeffect on LDL-proteoglycan binding, preferably inhibitory activity.Candidate compounds may be inorganic or organic, though typically theyare organic. Candidate compounds may be naturally occurring orsynthetic. Candidate compounds are typically pharmacologically active“small molecules”, but also include biological molecules such as aminoacids, proteins, glycoproteins, lipoproteins, saccharides,polysaccharides, lipopolysaccharides, fatty acids, and nucleic acids.Examples of organic candidate compounds also include antibodies,antigens, haptens, enzymes, hormones, steroids, vitamins,oligonucleotides, and pharmacological agents.

The terms “sample” and “sample composition,” as used herein, pertain toa composition which comprises one or more agents or candidate compoundsof interest, or which may be processed to comprise one or more candidatecompounds of interest. The samples used can be defined combinatoriallibraries or undefined biological samples (e.g. crude plant extracts,and fungal broths). The sample or candidate compound may be in solid,emulsion, suspension, liquid, or gas form. Typically, the sample orcandidate compound is processed (e.g., by the addition of a liquidbuffer) so as to be a fluid (i.e., free flowing) form (e.g., emulsion,suspension, solution) in order to readily permit and simplify thedetection and quantification of the LDL-proteoglycan binding in thecompound's presence using the methods of the invention. Typically, thesample or candidate compound of interest is present in the samplecomposition at a concentration of 10⁻³ M (micromolar) or less, forexample, often as low as 10⁻⁹ M (nanomolar), sometimes as low as 10⁻¹² M(picomolar), and even as low as 10⁻¹³ M (sub-picomolar).

The “LDL” reagent used in the assay can be any reagent which comprisesat least a fragment of apo-B100 protein and maintains theLDL-proteoglycan binding activity of at least 60% of wild-type levels,preferably at least 70%, more preferably at least 80%, still morepreferably 90%, most preferably at essentially 100% of wild-typeLDL-proteoglycan binding activity. The apo-B100 fragment is preferablycomplete and preferably a wild-type human apo-B100 protein, but mutantproteins which maintain proteoglycan binding activity can be employed.The LDL reagent can be an LDL expressed in a non-human animal or mammal,like the recombinant control LDL described in Examples 3 and 4.Preferably the “LDL” reagent used in the assays is normal human plasmaLDL obtained from human blood and purified as described in Example 4.The “LDL” used for the assay methods may optionally be labeled tofacilitate detection or measurement of LDL-proteoglycan complex formed.The LDL may be labeled by any means known in the art including theincorporation of radionuclides (e.g. ¹²⁵I, ³⁵S, etc.) into the proteinsor lipids of the LDL, inclusion of fluorescent lipid (e.g. diI), theattachment of enzymes (e.g. β-galactosidase, horseradish peroxidase,etc.), or the attachment of one of a pair of detectable binding partners(e.g. biotinylation).

The “proteoglycan” reagent used in the assay can be any proteoglycanwhich binds specifically to human LDL. Preferably proteoglycans isolatedfrom the artery wall of an animal, mammal, or human, or isolated fromarterial smooth muscle cells, preferably human, are used, includingversican, perlecan, biglycan, or decorin. In addition, any commerciallyavailable preparation of proteoglycan or glucosaminoglycan can be usedincluding chondroitin disaccharides, heparin, chondroitin sulfate A,chondroitin sulfate B, chondroitin sulfate C, heparin disaccharides,heparin-like substance sulodexide, or heparin-like substance mesoglycan.(Sigma) As with the LDL of the invention the “proteoglycan” reagent usedin the assay methods may optionally be labeled to facilitate detectionmeasurement of LDL-proteoglycan complex formed. The proteoglycan may belabeled by any means known in the art including the incorporation ofradionuclides (e.g. ¹²⁵I, ³⁵S, etc.) into the proteins or disaccharidesof the proteoglycan, the attachment of enzymes (e.g. β-galactosidase,horseradish peroxidase, etc.), or the attachment of one of a pair ofdetectable binding partners (e.g. biotinylation).

The assays of the invention involve mixing a proteoglycan reagent and anLDL reagent in the presence of a test compound, under conditions whereinthe LDL binds to the proteoglycan to form an LDL-proteoglycan complex inthe absence of said candidate compound. The appropriate conditions forsuch reaction mixtures are known in the art (See, e.g., Examples 8 and10), and can in addition be determined empirically by observing whetherLDL-proteoglycan complexes are formed. Protocols may utilize a solidsupport to separate unbound LDL and proteoglycan reagents fromLDL-proteoglycan complex, or this separation may be performed byimmunoprecipitation, separation by gel electrophoresis, or affinitychromatography.

In one preferred embodiment, the level of LDL-proteoglycan binding isdetermined by gel-mobility shift assay. Prior to the assay, radiolabeledproteoglycan preparations are dialyzed. Human plasma LDL and a candidatecompound are incubated with approximately 2000 dpm of (³⁵S)biglycan or(³⁵S)versican for 1 h at 37° C. The samples are loaded into wells onagarose gel, and subjected to electrophoresis. Gels are then fixed,dried, and exposed to film. The (³⁵S)biglycan or (³⁵S)versican complexedto LDL appears as a band at the origin of the can be quantitativelyevaluated. This procedure has the advantages that only microgramquantities of lipoproteins are required and the relative affinity of LDLbinding to the proteoglycans can be determined at physiological ionicand pH conditions.

In a second preferred embodiment proteoglycan-LDL binding is measured inthe presence of a candidate compound as a drug screening assay. Aprocedure for a competitive solid-phase plate assay is employed. Normalhuman plasma LDL (1.0 μg in 50 μl of phosphate-buffered saline (PBS)containing 0.01% EDTA per well) is immobilized by absorption to a solidsupport, preferably a polystyrene 96-well micrometer plates for 6 to 24hours at 4° C. Excess LDL is removed by washing in PBS, and nonspecificsites on the plastic are blocked by incubation with PBS containing 5%bovine serum albumin (BSA) for 1 to 24 hours at 24° C. The wells arewashed three times with PBS and then with binding buffer (10 mM Tris, 50mM NaCl, 5 mM CaCl₂, 0.05% BSA). Biotinylated proteoglycans along with acandidate compound, preferably in micromolar quantities, are added toeach well and incubated for approximately 1 hour at 24° C. The unboundproteoglycans are removed and the wells are washed for up to three timeswith 50 mM Tris, 90 mM NaCl, 5 mM CaCl₂, 0.05% BSA. Then 50 μl ofstreptavidin peroxidase (10 μg/ml) is added and incubated forapproximately 2 hours at 24° C. The unbound streptavidin peroxidase isremoved and the wells are washed three times with 50 mM Tris, 90 mMNaCl, 5 mM CaCl₂, 0.05% BSA. Finally a peroxidase substrate, preferablychromogen o-dianisidine, is added in an appropriate buffer andabsorbency at 405 nm is measured. Negative control values are obtainedby using normal human plasma LDL, or in its place recombinant LDLcomprising wild-type human apo-B100, obtained as described above inExamples 1-4. When the proteoglycans are added no candidate compound isadded to the negative control wells. Negative control values representnormal LDL proteoglycan binding. Positive control wells are obtainedusing the RK3359-3369SA LDL and the K3363E LDL obtained as describedabove in Examples 1-4 in place of the normal human plasma LDL. Again,when the proteoglycans are added no candidate compound added to thepositive control wells. Positive control values represent defective LDLproteoglycan binding. Those candidate compounds which reduceLDL-proteoglycan binding are identified for further testing and possibleuse as lead compounds for pharmaceutical development and use.

In particularly preferred embodiments the assays of the invention areperformed by robots which are able to add defined quantities of reagentsto the well of a plate, as well as perform washes and incubation stepsat various temperatures.

In another preferred embodiment candidate compounds which have beenshown to affect, particularly inhibit, LDL-proteoglycan binding aretested to see if they also affect LDL/LDL receptor binding. Thisembodiment is particularly useful in drug screening assays in whichcompounds that disrupt LDL-proteoglycan binding with out affectingLDL/LDL receptor binding are sought as lead compounds as a part of thedrug discovery process. In a particularly preferred embodiment mixturesof comprising LDL receptor, LDL, and a candidate compound that affectsLDL-proteoglycan binding, are incubated under conditions wherein LDLbinds to LDL receptor to form an LDL-LDL receptor complex in the absenceof said inhibitor of LDL-proteoglycan binding. The difference betweenthe amount of LDL-LDL receptor complex present in the mixture preparedwith the candidate compound, and a control mixture prepared without thecandidate compound are compared, and optionally any difference iscorrelated with said candidate compound's ability to affect LDL—LDLreceptor binding activity.

Transgenic Animals In Vivo Model for Atherosclerosis

In addition, the present invention encompasses the use of transgenicnon-human animals and mammals which express human apo-B100 as an in vivomodel system for the study of atherosclerosis, and in vivo assay methodsfor identifying compounds which modulate atherosclerosis and/orLDL-proteoglycan binding. Identifying compounds which inhibitatherosclerosis or LDL-proteoglycan binding activity is the preferreduse of this assay, but it can equally be used to identify compoundswhich result in an increase in atherosclerotic regions. Thus, the assaysof the invention may be used to determine whether a particular food ordrug composition tends to stimulate or inhibit the formation ofatherosclerotic lesions. The in vivo assay of the invention generallyinvolve administering a sample or candidate compound to the transgenicanimal, measuring the extent of atherosclerosis or atheroscleroticlesions which results, and correlating the measured extent ofatherosclerosis or atherosclerotic lesions with the candidate compound'sability to modulate atherosclerosis in vivo, typically by using arelationship determined from one or more control animals. In a preferredembodiment at least one of the control animals used expresses aproteoglycan⁻receptor⁺ LDL.

In another preferred embodiment the transgenic non-human animals ormammals to which the candidate compounds or samples are administered,express a human apo-B100 protein in which the glutamine at amino acidposition 2153 has been replaced with a leucine. This mutation abolishesthe formation of apo-B48, resulting in a higher yield of recombinantapo-B100 LDL,. Apo-B48 has distinct proteoglycan binding site(s) fromSite B in apo-B100. Therefore use of this mutation in the in vivo assaymethods of the invention provides an effective means for studying theportion of atherogenesis which is the result of apo-B100 mediatedLDL-proteoglycan binding, as opposed to apo-B48 mediated chylomicron-and chylomicron remnant-proteoglycan binding.

In a qualitative assay, simply determining whether the measuredatherosclerosis or atherosclerotic lesions is above or below a thresholdvalue (established, for example, using recombinant LDL samples withknown LDL-proteoglycan binding properties) may be sufficient toestablish the assay result. Thus, unless otherwise required, the term“measuring” can refer to either qualitative or quantitativedetermination.

Typically, the sample or candidate compound described above for theassay for identifying agents affecting of LDL-proteoglycan binding isadministered to the non-human mammal or animal, preferably a candidatecompound which has demonstrated inhibition of LDL-proteoglycan bindingin vitro is used. Preferably, the sample or candidate compound haspreviously been identified as a compound which affects or inhibitsLDL-proteoglycan binding in one of the assays of the invention. Thecandidate compound can be administered by any means known in the art.Typically, the amount of sample or candidate compound of interest iscontrolled to deliver in a dose of 10⁻³ M (micromolar) or less, forexample, often as low as 10⁻⁹ M (nanomolar), and sometimes as low as10⁻¹² M (picomolar) in the subject animal's plasma.

The animal used can be any non-human animal preferably a mammal, morepreferably a primate, rabbit, pig, goat or rodent, still more preferablya mouse, most preferably the recombinant control mouse described inExample 3. The animal must express a human apo-B100 protein, or at leasta sufficiently large fragment thereof to allow the animal's recombinantLDL to bind to endogenous arterial wall, which can be predicted bydemonstrating LDL-proteoglycan binding in an in vitro assay (See, e.g.,Example 8).

In addition, the non-human transgenic animal must be subjected toconditions wherein measurable atherosclerotic lesions form in thearteries of said animal. As used herein the phrase “conditions whereinmeasurable atherosclerotic lesions form in the arteries of said animal”is used to denote any conditions which are known to causeatherosclerotic lesions in the particular animal used in an experiment.These conditions are particular to the animal used and must bedetermined empirically to ensure that the lesions are in fact measurableby whatever method is used. Most of such conditions relate to the dietof the animal or to the animals genetic make up. With respect to diet,cholesterol, cholesteryl ester, bile salts and fats, particularlysaturated fats are known to induce atherosclerotic lesions when consumedin high doses. The Paigen diet described in Example 9 is an example ofsuch a dietary condition. In terms of genetics, factors such asdefective LDL receptors, mutant apolipoprotein genes, particularlyapo-B100, which disrupt LDL/LDL receptor binding are example of geneticconditions.

The amount of atherosclerosis or atherosclerotic lesions is measured bymany methods known in the art including the morphometric imaging methoddescribed in Example 9, as well as arteriography and ultrasound. Thedifference between the extent of atherosclerosis or atheroscleroticlesions present in the animal which has been administered the testcompound and a control animal which has not received the test compoundis determined. Preferably the control animal is precisely the same typeand strain of animal and that which received the candidate compound, andhas been treated with the same conditions.

Mutant apo-B100 Proteins, Fragments and LDL

The invention embodies polypeptides comprising a proteoglycan⁻receptor⁺Site B, and entire apo-B100 proteins comprising a proteoglycan⁻receptor⁺Site B, as well as fragments thereof which comprise aproteoglycan⁻receptor⁺ Site B flanked on at least one side by acontiguous sequence of at least N amino acids which is directly adjacentto Site B in the wild-type human apo-B100 sequence, where the number Nis about 25 amino acids, preferably 20 amino acids, more preferably 15amino acid, still more preferably 8 to 10 amino acids, most preferably 6amino acids. The apo-B100 proteins of the invention also compriseproteins that have a leucine in place of glutamine at amino acidposition 2153, which abolishes the formation of apo-B48, resulting in ahigher yield of recombinant apo-B100 LDL.

The proteins of the invention can be made using routine expressionmethods known in the art. The DNA encoding the desired polypeptide, maybe ligated into an expression vector suitable for any convenient host.Both eukaryotic and prokaryotic host systems may be used in formingrecombinant polypeptides, and a summary of some of the more commonsystems are included below in the description of expression vectors. Thepolypeptide is then isolated from lysed cells or from the culture mediumand purified to the extent needed for its intended use. Purification maybe by any technique known in the art, for example, differentialextraction, salt fractionation, chromatography, centrifugation, and thelike. See, for example, Methods in Enzymology for a variety of methodsfor purifying proteins.

In addition, shorter protein fragments may be produced by chemicalsynthesis. Alternatively the proteins of the invention may be extractedfrom recombinant LDL. Methods for purifying apolipoproteins,particularly apo-B100 are known in the art, and include the use ofdetergents or chaotropic agents to disrupt particles followed bydifferential extraction and separation of the apo-B100 proteins andlipids by ion exchange chromatography, affinity chromatography,sedimentation according to density, and gel electrophoresis. RecombinantLDL can be isolated from a transgenic animal as described in Examples 3and 4. The term recombinant LDL also embraces reconstituted LDL as wellas LDL derived from a transgenic non-human animal, as described above.Methods of reconstituting LDL are known in the art. See, for example,Corsini, A. et al. 1987. J. Lipid Res. 28:1410-1423. Such reconstitutedLDL may comprise lipids from solely human sources, as well as lipids andlabels which are not naturally associated with LDL. Such reconstitutedrecombinant LDL must, however, comprise a mutant human apo-B100 proteinof the invention.

As used herein, the term “purified recombinant LDL” refers to arecombinant LDL which is essentially free, i.e., contains less thanabout 50%, preferably less than about 30%, and more preferably less thanabout 10%, even more preferably less than about 5%, and still morepreferably less than about 1% of lipoproteins comprising one or morenon-human apolipoproteins. Methods for purifying recombinant LDL includecentrifugation to separate lipoproteins of a particular density fromother plasma constituents, as well as affinity chromatography utilizingan antibody which is specific for an antigenic determinant found only onthe recombinant LDL or only on the other lipoproteins produced by atransgenic non-human animal.

The present invention also encompasses non-LDL lipoprotein particleswhich comprise an apo-B100 protein or fragment of the invention. Theselipoproteins may include other human apolipoproteins which are normallyassociated with apo-B100 (e.g. apo-E and possibly apo-C), and may havethe same rough physical properties of VLDL or IDL. They may also includeapo-lipoproteins which are native to the transgenic animal from whichthey are isolated. These non-LDL lipoproteins particles can be isolatedand used as a source of purified mutant apo-B100 protein.

Antibodies to Proteoglycan⁻Receptor⁺ Mutant apo-B100 Proteins and LDL

Apo-B100 proteins comprising proteoglycan⁻receptor⁺ mutations, fragmentsthereof comprising Site B, and recombinant LDL particles comprisingeither of these apo-B100 proteins or fragments are used to produceantibodies, including both polyclonal and monoclonal. If polyclonalantibodies are desired, a suitable non-human animal, preferably anon-human mammal is selected, usually a mouse, rat, rabbit, goat, orhorse, is immunized with an apo-B100 protein, fragment, or LDLcomprising the proteoglycan⁻receptor⁺ Site B in the presence of anappropriate adjuvant (e.g. aluminum hydroxide, RIBI, etc.) which isknown in the art. In addition the protein, fragment or LDL can bepretreated with an agent which will increase antigenicity, such agentsare known in the art and include, for example, methylated bovine serumalbumin (mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen,and keyhole limpet hemocyanin (KLH). Serum from the immunized animal iscollected, treated and tested according to known procedures. If theserum contains polyclonal antibodies to undesired epitopes, thepolyclonal antibodies can be purified by immunoaffinity chromatography.Techniques for producing and processing polyclonal antisera are known inthe art, see for example, Mayer and Walker (1987).

Alternatively, monoclonal antibodies directed against an apo-B100protein, fragment, or LDL comprising the proteoglycan⁻receptor⁺ Site Bcan also be readily produced by one of ordinary skill in the art. Thegeneral methodology for making monoclonal antibodies by hybridoma iswell known. Immortal antibody-producing cell lines can be created bycell fusion. See, for example, Harlow, E., and D. Lane. 1988. AntibodiesA Laboratory Manual. Cold Spring Harbor Laboratory. pp. 53-242.

Transgenic non-human animals or mammals which express a human apo-B100LDL with a wild-type Site B amino acid sequence are particularly usefulfor preparing antibodies, as these animals will recognize all or most ofthe exposed regions of human apo-B100 as self antigens. Thus, when suchan animal is exposed, for example, to an LDL particle comprising anapo-B100 with a proteoglycan⁻receptor⁺ Site B, this Site B will be oneof the few if not the only new immunogenic site exposed, and antibodiesto this mutant site will be preferentially produced. Alternatively, theantibodies of the invention can be screened by standard ELISA techniquefor there ability to bind to apo-B100 with a proteoglycan⁻receptor⁺ SiteB, while not binding to a recombinant control LDL.

Antibodies, both monoclonal and polyclonal, which are directed to aproteoglycan⁻receptor⁺ Site B are useful for screening for the presenceof such mutations in the population at large with standard RIA and ELISAassay techniques. In addition these antibodies are may be used to purifythe a proteoglycan⁻receptor⁺ recombinant LDL by affinity chromatography.

Polynucleotides, Cells, and Transgenic Animals

The invention embodies polynucleotides which encode a polypeptidecomprising a proteoglycan⁻receptor⁺ Site B, and entire apo-B100 proteinscomprising a proteoglycan⁻receptor⁺ Site B, as well as fragments thereofwhich comprise a proteoglycan⁻receptor⁺ Site B flanked on at least oneside by a contiguous sequence of at least N amino acids which isdirectly adjacent to Site B in the wild-type human apo-B100 sequence,where the number N is about 25 amino acids, preferably 20 amino acids,more preferably 15 amino acids, still more preferably 8 to 10 aminoacids, most preferably 6 amino acids. Generally the polynucleotides ofthe invention comprise the naturally occurring nucleotide sequence forthe portions of the gene which encode the amino acid sequences outsideof Site B as shown in the apo-B100 gene sequence of Knott, T, et al.1986. Nature 323:734-738. However, any naturally occurring silent codonvariation or other silent codon variation can be employed to encodethose amino acids outside of Site B. Similarly those nucleotidesequences which encode the portions of Site B which maintain thewild-type apo-B100 sequence will generally make use of the naturallyoccurring nucleotide sequence, but any naturally occurring silent codonvariation or other silent codon variation can be employed. As for thoseamino acids which are changed or added to the proteoglycan⁻receptor⁺Site B, nucleic acid sequences generally will be chosen to optimizeexpression in the specific human or non-human animal in which thepolynucleotide is intended to be used, making use of known codonpreferences.

The nucleic acids of the invention include expression vectors,amplification vectors, PCR-suitable polynucleotides, and vectors whichare suitable for the introduction of a polynucleotide of the inventioninto an embryonic stem cell for the production of transgenic non-humananimals. In addition, vectors which are suitable for the introduction ofa polynucleotide of the invention into cells, organs and individuals,including human individuals, for the purposes of gene therapy to reducethe severity of or prevent atherosclerosis are encompassed. Theinvention also encompasses targeting vectors and method for changing awild-type Site B into a proteoglyean⁻receptor⁺ Site B in a humanapo-B100 gene contained within an embryonic stem cell.

The invention embodies amplification vectors, which comprise apolynucleotide of the invention, and an origin of replication.Preferably, such amplification vectors further comprise restrictionendonuclease sites flanking the polynucleotide, so as to facilitatecleavage and purification of the polynucleotides from the remainder ofthe amplification vector, and a selectable marker, so as to facilitateamplification of the amplification vector. Most preferably, therestriction endonuclease sites in the amplification vector are situatedsuch that cleavage at those site would result in no other amplificationvector fragments of a similar size.

Thus, such an amplification vector may be transfected into a host cellcompatible with the origin of replication of said amplification vector,wherein the host cell is a prokaryotic or eukaryotic cell, preferably amammalian, insect, yeast, or bacterial cell, most preferably anEscherichia coli cell. The resulting transfected host cells may be grownby culture methods known in the art, preferably under selectioncompatible with the selectable marker (e.g., antibiotics). Theamplification vectors can be isolated and purified by methods known inthe art (e.g., standard plasmid prep procedures). The polynucleotide ofthe invention can be cleaved with restriction enzymes that specificallycleave at the restriction endonuclease sites flanking thepolynucleotide, and the double-stranded polynucleotide fragment purifiedby techniques known in the art, including gel electrophoresis.

Alternatively linear polynucleotides comprising a polynucleotide of theinvention may be amplified by PCR. The PCR method is well known in theart and described in, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 andSaiki, R et al. 1988. Science 239:487-491, and European patentapplications 86302298.4, 86302299.2 and 87300203.4, as well as Methodsin Enzymology 1987 155:335-350.

The polynucleotides of the invention also include expression vectors.Expression vector systems, control sequences and compatible host areknown in the art. For a review of these systems see, for example, U.S.Pat. No. 5,350,671, columns 45-48.

The polynucleotides of the invention can also be derivatized in variousways, including those appropriate for facilitating transfection and/orgene therapy. The polynucleotides can be derivatized by attaching anuclear localization signal to it to improve targeted delivery to thenucleus. One well-characterized nuclear localization signal is theheptapeptide PKKKRKV (pro-lys-lys-lys-arg-lys-val) (SEQ ID NO:21).Preferably, in the case of polynucleotides in the form of a closedcircle, the nuclear localization signal is attached via a modified loopnucleotide or spacer that forms a branching structure.

If it is to be used in vivo, the polynucleotide of the invention may bederivatized to include ligands and/or delivery vehicles which providedispersion through the blood, targeting to specific cell types, orpermit easier transit of cellular barriers. Thus, the polynucleotides ofthe invention may be linked or combined with any targeting or deliveryagent known in the art, including but not limited to, cell penetrationenhancers, lipofectin, liposomes, dendrimers, DNA intercalators, andnanoparticles. In particular, nanoparticles for use in the delivery ofthe polynucleotides of the invention are particles of less than about 50nanometers diameter, nontoxic, non-antigenic, and comprised of albuminand surfactant, or iron as in the nanoparticle particle technology ofSynGenix. In general the delivery vehicles used to target thepolynucleotides of the invention may further comprise any cell specificor general targeting agents known in the art, and will have a specifictrapping efficiency to the target cells or organs of from about 5 toabout 35%.

The polynucleotides of the invention may be used ex vivo in a genetherapy method for obtaining cells or organs which produceproteoglycan⁻receptor⁺ LDL. The cells are created by incubation of thetarget cell with one or more of the above-described polynucleotidesunder standard conditions for uptake of nucleic acids, includingelectroporation or lipofection. In practicing an ex vivo method oftreating cells or organs, the concentration of polynucleotides of theinvention in a solution prepare to treat target cells or organs is fromabout 0.1 to about 100 μM, preferably 0.5 to 50 μM, most preferably from1 to 10 μM.

Alternatively, the oligonucleotides can be modified or co-administeredfor targeted delivery to the nucleus. Improved oligonucleotide stabilityis expected in the nucleus due to: (1) lower levels of DNases andRNases; and (2) higher oligonucleotide concentrations due to lower totalvolume.

Alternatively, the polynucleotides of the invention can be covalentlybonded to biotin to form a biotin-polynucleotide prodrug by methodsknown in the art, and co-administered with a receptor ligand bound toavidin or receptor specific antibody bound to avidin, wherein thereceptor is capable of causing uptake of the resultingpolynucleotide-biotin-avidin complex into the cells. Receptors thatcause uptake are known to those of skill in the art. Any transplantablecell type or organ can be used preferably hepatic cells, fetal hepaticcells or whole or partial livers.

The invention encompasses vectors which are suitable for theintroduction of a polynucleotide of the invention into an embryonic stemcell for the production of transgenic non-human animals, which in turnresult in the expression of recombinant LDL in the transgenic animal.The size of the apo-B100 dictates that a vector which can accommodateinserts which are tens of thousands of bases long. Yeast artificialchromosomes (YAC), bacterial artificial chromosomes (BAC), bacteriophageP1, and other vectors known in the art which are able to accommodatesufficiently large inserts to encode the entire apo-B100 gene. The 95-kbapo-B P1 plasmid p158 of Linton, M. et al. 1993. J. Clin. Invest.92:3029-3037 makes a convenient vector system to use, as it alreadycontains a full length apo-B100 gene.

Moreover, Borén, J. et al. 1996. Genome Res. 6:1123-1130 havedemonstrated how to isolate a 5.7 kb fragment of the apo-B100 gene whichcomprises Site B, in order to perform site-directed mutagenesis asdescribed below in Examples 1 and 2, using RARE cleavage. In brief,RecA-assisted restriction endonuclease (RARE) cleavage consists ofprotecting a specific restriction endonuclease site with a complementaryoligonucleotide. In the presence of RecA, a triplex DNA complex isformed that prevents methylation at the protected sites, for exampleEcoRI-35763 and EcoRI-41496 were protected by oligonucleotides (5′gaaaactcccacagcaagctaatgattatctgaattcattcaattgggagagacaa gtttcac 3′)(SEQ ID NO:22) and (5′cacaagtgaaatatctggttaggatagaattctcccagttttcacaatgaaaacatc 3′) (SEQ IDNO:23) respectively, while unprotected sites are methylated by thecorresponding methylase. After dissociation of the oligonucleotides, theprotected sites can be cleaved with the restriction endonuclease whichcorresponds to the protected sites, for example EcoRI. All of thenon-protected EcoRI site had been methylated and were thus not subjectto cleavage by the restriction enzyme. The resulting fragment of theapo-B100 gene can then be ligated into a smaller vector which isappropriate for site-directed mutagenesis, e.g. pZErO. The site-directedmutagenesis process is then conducted by techniques well known in theart, and the fragment is return and ligated to the larger vector fromwhich it was cleaved. For site directed mutagenesis methods see, forexample, Kunkel, T. 1985. Proc. Natl. Acad. Sci. U.S.A. 82:488;Bandeyar, M. et al. 1988. Gene 65:129-133; Nelson, M., and M. McClelland1992. Methods Enzymol. 216:279-303; Weiner, M. 1994. Gene 151:119-123;Costa, G. and M. Weiner. 1994. Nucleic Acids Res. 22:2423; Hu, G. 1993.DNA and Cell Biology 12:763-770; and Deng, W. and J. Nickoff. 1992.Anal. Biochem. 200:81.

Briefly, the transgenic technology used herein involves theinactivation, addition or replacement of a portion of a gene or anentire gene. For example the present technology includes the addition ofhuman proteoglycan⁻receptor⁺ apo-B100 genes with or without theinactivation of the non-human animal's native apolipoprotein genes, asdescribed in the preceding two paragraphs and in the Examples. Theinvention also encompasses the use of vectors, and the vectorsthemselves which target and modify an existing human apo-B100 gene in astem cell, whether it is contained in a non-human animal cell where itwas previously introduced into the germ line by transgenic technology orit is a native apo-B100 gene in a human pluripotent cell. This transgenetechnology usually relies on homologous recombination in a pluripotentcell that is capable of differentiating into germ cell tissue. A DNAconstruct that encodes an altered region of comprising aproteoglycan⁻receptor⁺ Site B or an altered region of the non-humananimal's apolipoprotein gene the contains, for instance a stop codon todestroy expression, is introduced into the nuclei of embryonic stemcells. Preferably mice are used for this transgenic work. In a portionof the cells, the introduced DNA recombines with the endogenous copy ofthe cell's gene, replacing it with the altered copy. Cells containingthe newly engineered genetic alteration are injected in a host embryo ofthe same species as the stem cell, and the embryo is reimplanted into arecipient female. Some of these embryos develop into chimericindividuals that posses germ cells entirely derived from the mutant cellline. Therefore, by breeding the chimeric progeny it is possible toobtain a new strain containing the introduced genetic alteration. SeeCapecchi 1989. Science. 244:1288-1292 for a review of this procedure.

The present invention encompasses the polynucleotides described herein,as well as the methods for making these polynucleotides including themethod for creating a mutation in a proteoglycan⁻receptor⁺ mutation in ahuman apo-B100 gene. In addition, the present invention encompassescells which comprise the polynucleotides of the invention, including butnot limited to amplification host cells comprising amplification vectorsof the invention. Furthermore the present invention comprises theembryonic stem cells and transgenic non-human animals and mammalsdescribed herein which comprise a gene encoding a proteoglycan⁻receptor⁺human apo-B100 protein.

The invention also encompasses methods for preventing or reducing theseverity of atherosclerosis in an animal or mammal, by expressing apolynucleotide encoding a proteoglycan⁻receptor⁺ human apo-B100 proteinor protein fragment of the invention. The polynucleotide encoding aproteoglycan⁻receptor⁺ human apo-B100 protein or protein fragment of theinvention is transduced into a cell, either ex vivo or in situ. In thecase of ex vivo transduction, the transduced cell is then administeredto an animal or mammal. Expression of the proteoglycan⁻receptor⁺ humanapo-B100 protein or protein fragment of the invention substantiallyprevents, ameliorates or reduces the severity of atherosclerosis in theanimal or mammal.

The polynucleotide to be transduced is normally inserted into anappropriate expression vector, using standard molecular biologytechniques. Appropriate expression vectors are easily selected by one ofskill in the art, and generally include cis-acting transcription andtranslation nucleotide sequences which are operable in the cell to betransduced. Such elements are well known in the art, and include viralpromoters and enhancers (e.g., the SV40 promoter and enhancer),mammalian constituitive promoters (e.g., the β-actin promoter),mammalian tissue-specific promoters and enhancers, polyadenylationsignals, and the like. Preferably, an intron is introduced into thepolynucleotide encoding the proteoglycan⁻receptor⁺ human apo-B100protein or protein fragment, as the presence of an intron frequentlyimproves mRNA processing and export from the nucleus.

The expression vector may optionally include positive and negativeselection markers. Positive selection markers are preferable whentransduction is carried out ex vivo, because they permit enrichment ofcells transduced with the polynucleotide. Positive selection markers arewell known in the art, and include the neo^(r) and hyg^(r) genes, whichconfer resistance to gentamycin and hygromycin, respectively. A negativeselection marker may be included to allow termination of expression ofthe proteoglycan⁻receptor⁺ human apo-B100 protein or protein fragment,by killing of the transduced cells. One preferred negative selectionmarker is the herpes simplex virus 1 thymidine kinase (HSVtk) gene,which renders the transduced cells susceptible to ganciclovir.Alternately, fused positive/negative selection markers may be employed,such as the HyTK (hyg^(r)/HSVtk) fusion gene, which confers bothhygromycin resistance and ganciclovir sensitivity.

The expression construct may be transduced into the target cell usingany method known in the art, such as viral transduction,electroporation, lipid-mediated transduction, ballistic transduction,calcium phosphate transduction, or by naked DNA transfer, although viraltransduction, lipid-mediated transduction and naked DNA transfer arepreferred for in vivo transduction. In the case where viral transductionis employed, expression construct will also encode certain DNA or RNAvirus proteins and/or signals, to allow packaging into infectious viralparticles. The large size of the proteoglycan⁻receptor⁺ human apo-B100protein gene will constrain the selection of viral vectors for use intransducing target cells, as will be apparent to one of skill in theart, but most proteoglycan⁻receptor⁺ human apo-B100 protein fragmentconstructs can be inserted into any viral vector known to be suitablefor target cell transduction.

The quantity of cells transferred to the animal or mammal subject willdepend on a variety of factors, including the severity of the subject'satherosclerosis (or risk for developing atherosclerosis), the expressionlevels of the transduced cells, the subject's medical history, and otherfactors known to those of skill in the art. In any case, an effectiveamount of transduced cells (i.e., an amount sufficient to prevent,ameliorate, or reduce atherosclerosis in the subject) are transferred tothe subject.

Methods of ex vivo transduction are well known in the art. See, forexample, U.S. Pat. No. 5,399,346. Viral transduction is also well known,and is disclosed in a number of issued U.S. patents, such as U.S. Pat.Nos. 5,672,344, 5,656,465, 5,139,941, and 5,851,529. Transduction oftarget cells with naked DNA technology is disclosed in, for example,U.S. Pat. No. 5,580,859. The quantity of the polynucleotide of theinvention administered to the animal or mammal subject by in situtransduction will vary according to a number of parameters, such as theefficiency of the transduction method, the desired levels of expression,the subject's medical history, and other parameters known to one ofskill in the art. In any case, an effective amount of a polynucleotideof the invention (i.e., an amount sufficient to prevent, ameliorate, orreduce atherosclerosis in the subject) is administered to the subject.

As will be apparent to one of skill in the art, blood levels ofproteoglycan⁻receptor⁺ human apo-B100 protein or protein fragment may bemeasured after in situ transduction or ex vivo transduction and transferof transduced cells to the subject. If blood levels of the protein orprotein fragment are not at desired levels, transduction or transductionand transfer may be repeated to achieve the desired levels of theprotein or protein fragment of the invention.

After transduction (for in situ transduction) or transduction andtransfer to the animal or mammal (for ex vivo transduction), expressionof the polynucleotide encoding a proteoglycan⁻receptor⁺ human apo-B100protein or protein fragment results in prevention of or a reduction oramelioration of the severity of symptoms of atherosclerosis.

Throughout this application, various publications, patents, andpublished patent applications are cited. The disclosures of thepublications, patents, and published patent specifications referenced inthis application are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

EXAMPLES

Several of the methods of the present invention are described in thefollowing examples, which are offered by way of illustration and not byway of limitation. Many other modifications and variations of theinvention as herein set forth can be made without departing from thespirit and scope thereof and therefore only such limitations should beimposed as are indicated by the appended claims.

Example 1 Generation of Truncated P1 Plasmids and Isolation of DNAFragments for Mutagenesis

The 95-kb apo-B P1 plasmid p158 (Linton, M. et al. 1993. J. Clin.Invest. 92:3029-3037) was prepared and modified by RARE cleavage asdescribed by Borén, J. et al. 1996. Genome Res. 6:1123-1130. OligomersEcoRI-35763 (5′gaaaactcccacagcaagctaatgattatctgaattcattcaattgggagagacaagtttcac 3′) (SEQID NO:22) and EcoRI-41496 (5′cacaagtgaaatatctggttaggatagaattctcccagttttcacaatgaaaacatc 3′) (SEQ IDNO:23) were used to make 5.7-kb-deleted P1 plasmid. A 5.7-kb fragmentwas isolated from the a po-B100 “Leu—Leu” plasmid with RARE cleavageusing oligomers EcoRI-35763 and EcoRI-41496 and cloned into the pZErO-1vector (Invitrogen). The apo-B100 “Leu-Leu” plasmid was used to increasethe yield of apo-B100, since it contains a CAA to CTA mutation in codon2153 that effectively abolished the formation of apo-B48. The latter ofwhich is formed by an editing mechanism present in mouse livers (Yao, Z.et al. 1992. J. Biol Chem. 267:1175-1182).

Example 2 Site-Directed Mutagenesis of P1 DNA

The pZErO-5.7 kb plasmid was subjected to site-directed mutagenesis withthe Morph System (5 Prime→3 Prime, Inc.®) using oligonucleotide K3363E(5′ caagattgacaagagaaaggggattgaag 3′) (SEQ ID NO:24) to mutate thelysine at reside 3363 to glutamic acid. The resulting plasmids weresubjected to RARE cleavage with oligomers EcoRI-35763 and EcoRI-41496,and the mutated 5.7-kb fragment was isolated. After RARE cleavage of the5.7-kb-deleted P1 plasmid with oligonucleotide EcoRI del. 5.7-kb (5′ggaaaactcccacagcaagctaatgattatctgaattctccc agttttcacaatgaaaacatc 3′)(SEQ ID NO:25), the mutated 5.7-kb fragment was ligated into thelinearized and phosphatased 5.7-kb-deleted P1 vector (Borén, J. et al.1996. Genome Res. 6:1123-1130).

Example 3 Human Apo-B Transgenic Mice

The transgenic mice were generated with a P1 bacteriophage clone (p158)(Linton, M. et al. 1993. J. Clin. Invest. 92:3029-3037) that spanned thehuman apo-B gene in which mutations had been introduced by RecA-assistedrestriction endonuclease (RARE) cleavage (Borén, J. et al. 1996. GenomeRes. 6:1123-1130) as described in Examples 1 and 2.

P1 DNA was prepared and microinjected into fertilized mouse eggs(C57BL/6×SJL) (McCormick, S. et al. 1994. Genet Anal Tech Appl11:158-164). Mice were housed in a pathogen-free barrier facilityoperating on a 12-h light/12-h dark cycle and were fed rodent chowcontaining 4.5% fat (Ralston Purina, St. Louis, Mo.).

Transgenic mice were identified at the time of weaning (21 days) byscreening mouse plasma for human apo-B100 western dot-blot and westernanalysis with the monoclonal antibody 1D1 (Milne, R. et al. 1983.Arteriosclerosis. 3:23-30). Four different types of human recombinantLDL were generated (Table 1). The first of the transgenic mouse linesexpressed recombinant control LDL. This LDL, however, had a leucine inplace of glutamine at amino acid position 2153, which abolishes theformation of apo-B48, resulting in a higher yield of recombinantapo-B100 LDL. Its receptor-binding activity was found to be identical tothat of LDL generated by the unmodified apo-B100 P1 bacteriophage clone.The second transgenic mouse line expressed a form of recombinant LDLthat had a single amino acid mutation, the substitution of glutamine forthe normally occurring arginine at residue 3500 in apo-B100 (R3500Q). Wehave also proven that this mutation is identical to the mutation thatcauses defective receptor binding in the genetic disorder familialdefective apo-B 100 (Borén, J. et al. 1998. J. Clin. Invest.101:1084-1093). Although it is outside the receptor-binding site (SiteB), this mutation produces a conformational change that disruptsreceptor binding. This is the only LDL that did not have the “Leu—Leu”mutation encoded in the apo-B mRNA. The third transgenic mouse lineexpressed a recombinant LDL in which the basic amino acids in Site B(residues 3359-3369) were converted to neutral amino acids. The arginineresidues were converted to serines and the lysine residues to alanines(RK3359-3369SA). These changes virtually abolish the receptor-bindingactivity of the recombinant LDL; this finding along with other evidencedemonstrated that Site B is the receptor-binding site of LDL (Borén, J.et al. 1998. J. Clin. Invest. 101:1084-1093). The fourth transgenicmouse line expressed human recombinant LDL in which the lysine atresidue 3363 of apo-B100 was changed to glutamic acid (K3363E). Thismutation was designed to disrupt proteoglycan binding if Site B plays asignificant role in binding to proteoglycans.

TABLE 1 Mutants of the Human Apo-B Gene Recombinant LDL LDL Receptorbinding Proteoglycan binding Control LDL Normal Normal R3500Q LDLDefective Normal RK3359-3369SA LDL Defective Defective K3363E LDL NormalDefective

Example 4 Isolation of Recombinant Lipoproteins

Blood from mice fasted for 5 h was collected by cardiac puncture intotubes containing EDTA (final concentration 1 mg/ml), and the plasma wasmixed with butylated hydroxytoluene (final concentration, 25 μM),phenylmethyl sulfonylfluoride (final concentration, 1 mM), and aprotinin(final concentration, 10 U/ml). The LDL (d=1.02-1.05 g/ml) were isolatedby sequential ultracentrifugation (Ti 70 rotor) and dialyzed against 150mM NaCl and 0.01% EDTA, pH 7.4, and the mouse apo-E and apo-B wereremoved by immunoaffinity chromatography. The d=1.02-1.05 g/ml fractionwas mixed with an equal volume (850 μl) of AffiGel Hz (BioRad) andincubated for 17 hours at 4° C. in a gently rocking tube filled withnitrogen. The AffiGel Hz (100 g) had previously been coupled with mouseapo-E or mouse apo-B rabbit immunoglobulins from 50 ml of antiserum.Lipoproteins used for receptor-binding experiments were isolated andassayed within 1 week. Human plasma LDL, isolated from a single blooddonor, were included as a control in each experiment.

The recombinant LDL were isolated from the human apo-B transgenic mouseplasma by ultracentrifugation, and the endogenous mouse apo-E- andapo-B-containing lipoproteins were removed by immunoaffinitychromatography. The purified lipoproteins were analyzed on western blotsof 3-15% polyacrylamide-SDS gels with ECL western blotting detectionreagents (Amersham). Purified recombinant LDL isolated from the plasmaof the four lines of transgenic mice contained intact apo-B100 withoutany visible contamination (FIG. 1A). Western blot analysis showed thatthe recombinant LDL from all four transgenic mouse lines bound to themonoclonal antibody 1D1 (FIG. 1B), whose epitope is between amino acids474 and 539 in human apo-B100 (Milne, R. et al. 1983. Arteriosclerosis.3:23-30) and that only the unpurified recombinant LDL reacted withpolyclonal antibodies against mouse apo-B and mouse apo-E (FIG. 1C andFIG. 1D, respectively), confirming that endogenous mouse apo-B and apo-Ehad been completely removed.

Example 5 Modification Of Recombinant LDL

To selectively modify arginines or lysines in apo-B100, recombinant LDLwere incubated with acetic anhydride or cyclohexadione, respectively.Acetylation of LDL was carried out as described by Basu, S. et al. 1976.Proc. Natl. Acad Sci. USA. 73:3178-3182. In short, with continuousstirring in ice water bath, recombinant LDL (0.5 mg) in 1.0 ml 0.15 MNaCl and 0.01% EDTA were mixed with 1.5 μl saturated sodium acetatesolution every 15 min over 1 hr. Cyclohexanedione modification of LDLwas performed as by Mahley, R. et al. 1977. J. Biol Chem 252:7279-7287.Recombinant LDL (0.5 mg) in 1 ml of 0.15 M NaCl and 0.01% EDTA was mixedwith 2 ml of 0.15 M 1,2-cyclohexanedione in 0.2 M sodium borate buffer(pH 8.1) and incubated at 35° C. for 2 h. The sample was then dialyzedfor 48 h against 0.15 M NaCl and 0.01% EDTA at 4° C.

Example 6 Cell Culture and Competitive Receptor Binding Assay

Human fibroblasts were cultured in Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal bovine serum. Seven days before eachexperiment, the fibroblasts were plated into 12-well cell culture dishes(22-mm diameter per well) at ˜12000 cells/well in the same medium. Twodays before each experiment, the cells were transferred to DMEMcontaining 10% human lipoprotein-deficient serum. Normal human¹²⁵I-labeled LDL (2 μg/ml) along with increasing concentrations ofunlabeled lipoproteins were added to the cells in DMEM containing 25 mMHEPES and 10% human lipoprotein-deficient serum. After a 3-h incubationat 4° C., the surface-bound radioactivity was determined. The amount ofunlabeled lipoproteins needed to compete 50% with ¹²⁵I-labeled LDL wasdetermined from an exponential decay curve-fitting model (Arnold, K. etal. 1992. Lipoprotein Analysis. A Practical Approach. C. A. Converse,and E. R. Skinner, editors. Oxford University Press, Oxford. 145-168).

To evaluate the receptor-binding activity of the recombinant LDL, LDLfrom each transgenic line were tested with an in vitro competitivereceptor-binding assay (FIG. 2). Recombinant LDL with the uncharged SiteB (RK3359-3369SA) or with the R3500Q mutation had defective receptorbinding (ED₅₀>20 μg/ml for both), a finding in agreement with otherresults obtained in our lab (Borén, J. et al. 1998. J. Clin. Invest.101:1084-1093). The K3363E LDL had normal receptor binding equivalent tothat of control LDL (ED₅₀ 2.4 and 2.3 μg/ml, respectively). Moreover,since LDL with the K3363E mutation retained LDL receptor-bindingactivity, these results also indicate that the mutation did not affectthe overall folding and stability of the protein.

Example 7 Biglycan And Versican Isolation

Biglycan and versican were prepared from cultured human arterial smoothmuscle cells metabolically labeled with (³⁵S)SO₄, as describedpreviously (Chang, Y. et al. 1983. J. Biol. Chem. 258:5679-5688).Briefly, cell medium was concentrated on DEAE-Sephacel minicolumnsequilibrated in 8 M urea, 0.25 M NaCl, and 0.5% CHAPS. The (³⁵S)labeledproteoglycans were eluted with 8 M urea, 3 M NaCl, and 0.5% CHAPS andapplied to a Sepharose CL-2B column equilibrated in 8 M urea and 0.5%CHAPS. Small aliquots of the resulting fractions were counted to providea profile of the separated material. The fractions were then combinedinto four pools: pool 1, K_(av)=0.2; pool 2, K_(av),=0.2-0.4; pool 3,K_(av),=0.4-0.55; and pool 4; K_(av),=0.55-0.8. Eluted material in eachpool was concentrated on Centricon-50 spin columns and dialyzed intoBuffer A used for binding assays. The bulk of the (³⁵S)SO₄ radioactivitywas present in pools 1 and 3. Western blot analyses showed that pool 1contained versican and was negative for perlecan, biglycan, and decorin.Pool 3 contained only biglycan and no immunoreactivity for perlecan,versican, or decorin. Only very small amounts of decorin were found inpool 4.

Example 8 Gel-Mobility Shift Assay

The interaction between LDL and biglycan or versican was investigated bya gel-mobility shift assay (Camejo, G. et al. 1993. J. Biol Chem.268:14131-14137). Before the assay, the (³⁵S)biglycan and (³⁵S)versicanpreparations were dialyzed extensively at 4° C. against 10 mM HEPES, 150mM NaCl, 5 mM CaCl₂, and 2 mM MgCl₂ (pH 7.4, Buffer A), and the proteinconcentrations were determined (BioRad Laboratories) with bovine gammaglobulin as the standard. Increasing concentrations of LDL wereincubated with approximately 2000 dpm of (³⁵S)biglycan or (³⁵S)versicanfor 1 h at 37° C. in a total volume of 20 μl of Buffer A. Threemicroliters of bromophenol blue:glycerol (1:1, v/v) was added to thesamples, and 20 μl was loaded into wells of 0.7% NuSieve (FMCBioProducts) agarose gels prepared on Gel-Bond film (FMC BioProducts).Electrophoresis was run for 3 h at 60V with recirculating buffer (10 mMHEPES, 2 mM CaCl₂, 4 mM MgCl₂ pH 7.2) in a cold room. Gels were fixedwith 0.1% cetyl pyridium chloride in 70% ethanol for 90 min, air-dried,and exposed to Hyper Film-MP (Amersham Life Sciences) at ⁻70° C. The(³⁵S)biglycan or (³⁵S)versican complexed to LDL appears as a band at theorigin of the were quantitatively evaluated with a Hewlett Packard ScanJet II cx and ImageQuant software (Molecular Dynamics).

Gel-Shift Analysis of Recombinant LDL with Versican and Biglycan

To determine the ability of the different recombinant LDL to interactwith proteoglycans, recombinant control, R3500Q, RK3359-3369SA, andK3363E LDL were isolated and subjected to gel-shift analysis (Camejo, G.et al. 1993. J. Biol Chem. 268:14131-14137). This procedure has theadvantages that only microgram quantities of lipoproteins are requiredand the relative affinity of LDL binding to the proteoglycans can bedetermined at physiological ionic and pH conditions. In threeindependent experiments, recombinant control LDL and R3500Q LDL bound(³⁵S)versican and (³⁵S)biglycan almost as efficiently as human plasmaLDL, but recombinant RK3359-3369SA and K3363E LDL had severely impairedbinding to both (³⁵S)versican and (³⁵S)biglycan (FIG. 3). Thus,mutations of basic amino acids in Site B dramatically reduced theability of apo-B100 to interact with proteoglycans. Of particularinterest was that recombinant K3363E LDL interacted defectively withboth versican and biglycan but had normal receptor binding. Recombinantcontrol LDL with and without the “Leu—Leu” mutation displayed identicalbinding to (³⁵S)versican and (³⁵S)biglycan (data not shown). Thus, thismutation does not affect the binding of LDL to proteoglycans.

Gel-Shift Analysis of Acetylated or Cyclohexanedione-ModifiedRecombinant LDL

Mutagenesis of Site B severely reduced its interaction with versican andbiglycan. To test the importance of the remaining clusters of basicamino acids for the interaction of LDL with versican or biglycan, theremaining arginines or lysines in apo-B100 were selectively modified toabolish the receptor-binding and heparin-binding activities of LDL.Recombinant LDL isolated from human apo-B transgenic mice expressingrecombinant control or RK3359-3369SA LDL were divided into threealiquots. Two aliquots were selectively modified with acetic anhydrideor cyclohexanedione to change the arginines and lysines, respectively.The ability of the modified recombinant control or RK3359-3369SA LDL tobind (³⁵S)biglycan or (³⁵S)versican was compared with that of unmodifiedrecombinant LDL by gel-shift analysis. Again, unmodified RK3359-3369SALDL had greatly reduced ability to interact with (³⁵S)biglycan or(³⁵S)versican (FIG. 4). Furthermore, the unmodified RK3359-3369SA LDLbound proteoglycans almost identically before and after chemicalmodification; only a minor difference was detected in the (³⁵S)biglycangel-shift assay (FIG. 4A). These data demonstrate that Site B is themost important functional site for interaction with proteoglycans andthat the seven other potential sites do not play a significant role.

Example 9 Initial In Vivo Atherosclerosis Studies UsingProteoglycan-Binding-Defective LDL

These studies were designed to determine if elevated levels ofproteoglycan-binding-defective LDL would be less atherogenic thansimilar levels of wild-type recombinant LDL. The RK3359-3369SA constructwas used to generate mice expressing the proteoglycan-binding-defectiveLDL. The apo-B transgenic mice used in this atherosclerosis study werehybrids of the genetic strains C57BL/6 (50%) and SJL (50%).Non-transgenic mice with the same genetic background were also includedin the study. The transgenic mice were fed a Paigen diet containing 1.2%cholesterol, 0.5% bile salts, and 20% fat for 17 weeks. The mice werethen sacrificed, and the aortas were perfusion fixed and analyzed withthe en face procedure, in which the entire aorta is pinned out flat,stained with Sudan IV, and analyzed with a morphometric image-analysissystem (Image-1/AT) to quantitate the extent of atherosclerosis.

In both groups of transgenic mice, the percentage of the vessel wallcovered by atherosclerotic lesions correlated with the plasmacholesterol level (FIG. 5). However, the extent of atherosclerosisdiffered dramatically between the groups. The transgenic mice expressingthe RK3359-3369SA LDL had strikingly less atherosclerosis than miceexpressing the wild-type recombinant LDL. It should be emphasized thatthe only difference between these two groups of transgenic mice is themutation of the apo-B gene in the one group that prevents the binding ofLDL to proteoglycans and to the LDL receptor. Representative aortas froma single wild-type recombinant LDL mouse, an RK3359-3369SA LDL mouse,and a non-transgenic mouse are shown in FIG. 6. The non-transgenic mousehad essentially no atherosclerosis, a finding that was consistent withthe analysis of nine other non-transgenic mice that had been on ahigh-fat, high-cholesterol diet for 17 weeks.

Example 10 Proteoglycan-LDL Binding Drug Screening Assay

A procedure for a competitive solid-phase plate assay is employed(Edwards, I. et al. 1993. J. Lipid Res. 34:1155-1163; and Steele, R. etal. 1987. Atherosclerosis. 65:51-62). Normal human plasma LDL (1.0 μg in50 μl of phosphate-buffered saline (PBS) containing 0.01% EDTA per well)is immobilized by absorption to polystyrene 96-well micrometer platesfor 18 hours at 4° C. Excess LDL is removed by washing in PBS, andnonspecific sites on the plastic are blocked by incubation with PBScontaining 5% bovine serum albumin (BSA) for 2 hours at 24° C. The wellsare washed three times with PBS and then with binding buffer (10 mMTris, 50 mM NaCl, 5 mM CaCl₂, 0.05% BSA). Biotinylated proteoglycansalong with a candidate compound are added to each well and incubated for1 hour at 24° C. The unbound proteoglycans are removed and the wells arewashed three times with 50 mM Tris, 90 mM NaCl, 5 mM CaCl₂, 0.05% BSAbefore 50 μl of streptavidin peroxidase (10 μg/ml) is added andincubated for 2 hours at 24° C. The unbound streptavidin peroxidase isremoved and the wells are washed three times with 50 mM Tris, 90 mMNaCl, 5 mM CaCl₂, 0.05% BSA. Finally the peroxidase substrate, chromogeno-dianisidine, is added and absorbency at 405 nm is measured.

Negative control values are obtained by using normal human plasma LDL,or in its place recombinant LDL comprising wild-type human apo-B100,obtained as described above in Examples 1-4. When the proteoglycans areadded no candidate compound is added to the negative control wells.Negative control values represent normal LDL proteoglycan binding.

Positive control wells are obtained using the RK3359-3369SA LDL and theK3363E LDL obtained as described above in Examples 1-4 in place of thenormal human plasma LDL. Again, when the proteoglycans are added nocandidate compound added to the positive control wells. Positive controlvalues represent defective LDL proteoglycan binding.

Those candidate compounds which reduce LDL-proteoglycan binding areidentified for further testing and possible use as lead compounds forpharmaceutical development and use.

Example 11 Drug Screening Receptor Binding Assay

Those candidate compounds which demonstrate disruption ofLDL-proteoglycan binding in Example 10 or other LDL-proteoglycan bindingassays are tested to ensure that they do not disrupt LDL receptorbinding.

Human fibroblasts are cultured in Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal bovine serum. Seven days before eachexperiment, the fibroblasts are plated into 12-well cell culture dishes(22-mm diameter per well) at ˜12000 cells/well in the same medium. Twodays before each experiment, the cells are transferred to DMEMcontaining 10% human lipoprotein-deficient serum. Normal human¹²⁵I-labeled LDL (2 μg/ml) along with a candidate compound which hasbeen shown to disrupt LDL proteoglycan binding is added to each well inDMEM containing 25 mM HEPES and 10% human lipoprotein-deficient serum.After a 3-h incubation at 4° C., the medium is removed, and washed threetimes with DMEM containing 25 mM HEPES and 10% humanlipoprotein-deficient serum. The surface-bound radioactivity isdetermined for each well.

Example 12 Use of apo-B Transgenic Mice as In Vivo Atherosclerosis ModelSystem for Determining the Efficacy of Candidate Compounds

The transgenic mice described above in Examples 1-3 are used to test theefficacy of candidate compounds at reducing atherosclerosis. Transgenicmice expressing the wild-type human apo-B100 are each administered acandidate compound for 17 weeks, and fed a Paigen diet containing 1.2%cholesterol, 0.5% bile salts, and 20% fat during this period. The miceare then sacrificed, and the aortas are perfusion fixed and analyzedwith the en face procedure, in which the entire aorta is pinned outflat, stained with Sudan IV, and analyzed with a morphometricimage-analysis system (Image-1/AT) to quantitate the extent ofatherosclerosis.

Negative control values are obtained using transgenic mice with thewild-type human apo-B100 which are not administered a candidate compoundbut are fed the Paigen diet. Positive control values are obtained usingtransgenic mice expressing the RK3359-3369SA apo-B100 and the K3363E LDLapo-B100, which can be obtained as described in Examples 1-3 above,which are fed the Paigen diet for 17 weeks.

1. An isolated and purified polynucleotide encoding an apo-B100 protein comprising a proteoglycan⁻receptor⁺ mutation in Site B, wherein Site B is equivalent to amino acids from about 3358 to about 3369 of the human apo-B100 protein and wherein the mutation comprises at least one amino acid substitution or deletion of at least one of Lys₃₃₆₃ or Arg₃₃₆₂.
 2. The polynucleotide encoding an apo-B100 protein according to claim 1, wherein said at least one amino acid substitution in site B is an amino acid a residue selected from the group consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr Trp, Cys, Met, Asn, Gln, Asp, and Glu.
 3. The polynucleotide encoding an apo-B100 protein according to claim 1, wherein said mutation in Site B comprises replacement of all of the arginine residues and lysine residues with neutral amino acid residues.
 4. The polynucleotide encoding an apo-B100 protein according to claim 3, wherein said arginine residues are replaced with serine residues and said lysine residues are replaced with alanine residues.
 5. The polynucleotide encoding an apo-B100 protein according to claim 1, wherein said amino acid sequence from position 3358 to 3367 is SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:14.
 6. The polynucleotide encoding an apo-B100 protein according to claim 1, wherein said mutation in Site B is at position 3363 and the lysine residue is replaced with a glutamic acid residue, and the amino acid sequence from position 3358 to 3367 is: Thr₃₃₅₈-Arg₃₃₅₉-Leu₃₃₆₀-Thr₃₃₆₁-Arg₃₃₆₂-Glu₃₃₆₃-Arg₃₃₆₄-Gly₃₃₆₅-Leu₃₃₆₆-Lys₃₃₆₇ (SEQ ID NO:1).
 7. An isolated and purified polynucleotide encoding an apo-B100 protein comprising a proteoglycan⁻receptor⁺ mutation in Site B, wherein Site B is equivalent to amino acids from about 3358 to about 3369 of the human apo-B100 protein and wherein the mutation comprises at least one amino acid addition to site B.
 8. The polynucleotide encoding an apo-B100 protein according to claim 7, wherein said mutation in Site B is an addition of a single amino acid between positions 3362 and
 3364. 9. The polynucleotide encoding an apo-B100 protein according to claim 7, wherein said at least one amino acid addition to site B is selected from the group consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Cys, Met, Asn, Gln, Asp, and Glu.
 10. The polynucleotide encoding an apo-B100 protein according to claim 7, wherein said amino acid sequence from position 3358 to 3367 is SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.
 11. An isolated and purified polynucleotide encoding an apo-B100 protein comprising a proteoglycan⁻receptor⁺ mutation in Site B, wherein Site B is equivalent to amino acids from about 3358 to about 3369 of the human apo-B100 protein and wherein the mutation comprises a deletion of amino acid Arg₃₃₅₉ or a substitution of amino acid Arg₃₃₅₉ by an amino acid selected from the group consisting of Gly, Ala, Val, Leu, Ile, Phe, Tyr, Trp, Cys, Met, Asn, Gln, Asp, and Glu.
 12. The polynucleotide encoding an apo-B100 protein according to claim 11, wherein said amino acid sequence from position 3358 to 3367 is SEQ ID NO:11 or SEQ ID NO:12.
 13. An isolated and purified polynucleotide encoding an apo-B100 protein comprising a proteoglycan⁻receptor⁺ mutation in Site B, wherein Site B is equivalent to amino acids from about 3358 to about 3369 of the human apo-B100 protein and wherein the mutation comprises at least one amino acid substitution or deletion of Arg₃₃₆₄, wherein said at least one amino acid substitution in site B is an amino acid a residue selected from the group consisting of Gly, Ala Val, Leu, Ile, Phe, Tyr, Trp, Cys, Met, Ans, Gln, Asp, and Glu.
 14. The polynucleotide encoding an apo-B100 protein according to claim 13, wherein said amino acid sequence from position 3358 to 3367 is SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:15. 